Anti-fgfr2 antibody

ABSTRACT

The present invention provides an antibody which binds to a fibroblast growth factor receptor.

TECHNICAL FIELD

The present invention relates to a novel antibody, a nucleotidecomprising a nucleotide sequence encoding the amino acid sequence of theantibody, a vector having an insert of the nucleotide, a cell comprisingthe nucleotide or the vector, a method for producing the antibody,comprising the step of culturing the cell, a pharmaceutical compositioncomprising the antibody, a composition for diagnosis comprising theantibody, a functional fragment of the antibody, a modified form of theantibody, etc.

BACKGROUND ART

Fibroblast growth factors (FGFs) are known to play an important role inembryogenesis, tissue homeostasis, and metabolism via FGF receptor(FGFR) signals (Non Patent Literature 1). In humans, 22 FGFs (FGF1 toFGF14 and FGF16 to FGF23) and 4 FGF receptors (FGFR1 to FGFR4;hereinafter, collectively referred to as “FGFRs”) having a tyrosinekinase domain are found. These FGFRs are each constituted by anextracellular region comprising a ligand binding site composed of 2 or 3immunoglobulin-like domains (IgD1 to IgD3), a single-pass transmembraneregion, and an intracellular region comprising the tyrosine kinasedomain. FGFR1, FGFR2, and FGFR3 each have two splicing variants calledIIIb and IIIc. These isoforms differ in the sequence of approximately 50amino acids in the latter half of IgD3 and exhibit distinctive tissuedistribution and ligand specificity. It is generally known that the IIIbisoform is expressed in epithelial cells, while the IIIc isoform isexpressed in mesenchymal cells. Upon binding of FGFs to FGFRs, theseFGFRs are dimerized and phosphorylated at their particular tyrosineresidues. This phenomenon promotes the recruiting of important adaptorproteins such as FGFR substrate 2α (FRS2α) and induces the activation ofmany signaling pathways including MAPK and PI3K/Akt pathways. As aresult, FGFs and their corresponding receptors control a wide range ofcell functions including growth, differentiation, migration, andsurvival.

The abnormal activation of FGFRs is known to participate in particulartypes of malignant tumor development in humans (Non Patent Literature 1and 2). Particularly, findings such as the overexpression of FGFR2 andits ligand, receptor mutations or gene amplification, and isoformswitching, have been made as to the association of FGFR2 signalabnormality with cancer. Specifically, a single nucleotide polymorphism(SNP) in intron 2 of the FGFR2 gene reportedly correlates with the riskof breast cancer progression caused by the high expression of FGFR2 (NonPatent Literature 3 and 4). Missense mutations that constitutivelyactivate FGFR2 have been reported in endometrial cancer, ovary cancer,breast cancer, lung cancer, and stomach cancer (Non Patent Literature 2,3, and 5). Also, the amplification or overexpression of the FGFR2 genehas been reported in stomach cancer and breast cancer (Non PatentLiterature 2, 3, and 5). In addition, class switch from FGFR2 IIIb toFGFR2 IIIc is also known to occur during the progression of prostatecancer or kidney cancer and correlate with poor prognosis (Non PatentLiterature 6 and 7).

As mentioned above, the association of FGFR2 overexpression or mutationsor switching from IIIb to IIIc, with many cancer types suggests thepossibility of FGFR2 as an excellent therapeutic target for cancer. Infact, monoclonal antibodies against FGFR2 have been obtained and areunder evaluation for their antitumor effects in preclinical trials inorder to reveal the role of FGFR2 in oncogenesis and determine thepossibility of FGFR2 as a therapeutic target for cancer (Non PatentLiterature 8 and 9). All of these antibodies have been shown to have aneutralizing effect that inhibits signaling derived from a ligand forFGFR2 IIIb. Unfortunately, there has been no report on a functionalantibody having effector effects such as ADCC or a neutralizing effecton IIIc.

CITATION LIST Non Patent Literature

-   Non Patent Literature 1: Eswarakumar, V. P., et al., J. Cytokine    Growth Factor Rev., April 2005, Vol. 16 (No. 2), p. 139-149,    published online on Feb. 1, 2005, Review-   Non Patent Literature 2: Turner, N. and Grose, R., Nat. Rev. Cancer,    February 2010, Vol. 10 (No. 2), p. 116-129, Review-   Non Patent Literature 3: Easton, D. F., et al., Nature, Jun. 28,    2007, Vol. 447 (No. 7148), p. 1087-1093-   Non Patent Literature 4: Hunter D J, et al., Nat. Genet., July 2007,    Vol. 39 (No. 7), p. 870-874, published online on May 27, 2007-   Non Patent Literature 5: Katoh, Y. and Katoh, M., Int. J. Mol. Med.,    March 2009, Vol. 23 (No. 3), p. 307-311, Review-   Non Patent Literature 6: Chaffer, C. L., et al., Differentiation,    November 2007, Vol. 75 (No. 9), p. 831-842, published online on Aug.    14, 2007, Review-   Non Patent Literature 7: Carstens, R. P., et al., Oncogene, Dec. 18,    1997, Vol. 15 (No. 25), p. 3059-3065-   Non Patent Literature 8: Zhao, W. M., et al., Clin. Cancer Res.,    Dec. 1, 2010, Vol. 16 (No. 23), p. 5750-5758, published online on    Jul. 29, 2010-   Non Patent Literature 9: Bai, A., et al., Cancer Res., Oct. 1, 2010,    Vol. 70 (No. 19), p. 7630-7639, published online on Aug. 13, 2010

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide an antibody againstFGFR2.

Another object of the present invention is to provide a pharmaceuticalcomposition, etc. comprising an anti-FGFR2 antibody having an anticancereffect.

An alternative object of the present invention includes a nucleotideencoding the amino acid sequence of the antibody, a vector having aninsert of the nucleotide, a cell comprising the nucleotide or thevector, a method for producing the antibody, comprising the step ofculturing the cell, etc.

A further alternative object of the present invention is to provide amethod for treating cancer using the antibody.

Solution to Problem

The present inventors have conducted diligent studies to attain theobjects and consequently completed the present invention by developing anovel anti-FGFR2 antibody and have found that the antibody has ananticancer effect.

The present invention relates to:

(1) An antibody or a functional fragment thereof, which has antibodydependent cellular cytotoxic activity and binds to a fibroblast growthfactor receptor (FGFR);(2) The antibody or functional fragment thereof according to (1),wherein the fibroblast growth factor receptor (FGFR) is human FGFR;(3) The antibody or functional fragment thereof according to (1) or (2),wherein the fibroblast growth factor receptor (FGFR) is FGFR2;(4) The antibody or functional fragment thereof according to any one of(1) to (3), wherein the antibody or functional fragment thereof binds tohuman fibroblast growth factor receptor 2 (human FGFR2) IIIb and/orhuman fibroblast growth factor receptor 2 (human FGFR2) IIIc;(5) The antibody or functional fragment thereof according to any one of(1) to (4), wherein the antibody or functional fragment thereof binds tohuman fibroblast growth factor receptor 2 (human FGFR2) IIIb and humanfibroblast growth factor receptor (human FGFR2) IIIc;(6) The antibody or functional fragment thereof according to any one of(1) to (5), wherein the antibody or functional fragment thereof binds toone or two or more immunoglobulin-like domains of the human fibroblastgrowth factor receptor 2;(7) The antibody or functional fragment thereof according to any one of(1) to (6), wherein the antibody or functional fragment thereof binds toimmunoglobulin-like domain 2 of the human fibroblast growth factorreceptor 2;(8) The antibody or functional fragment thereof according to any one of(1) to (4) and (6), wherein the antibody or functional fragment thereofbinds to immunoglobulin-like domain 3 of the human fibroblast growthfactor receptor 2;(9) The antibody or functional fragment thereof according to any one of(1) to (8), wherein the antibody or functional fragment thereof hasneutralizing activity against the human fibroblast growth factorreceptor 2 (human FGFR2) IIIb and/or the human fibroblast growth factorreceptor 2 (human FGFR2) IIIc;(10) The antibody or functional fragment thereof according to any one of(1) to (9), wherein the antibody or functional fragment thereof hasneutralizing activity against the human fibroblast growth factorreceptor 2 (human FGFR2) IIIb and the human fibroblast growth factorreceptor 2 (human FGFR2) IIIc;(11) The antibody or functional fragment thereof according to any one of(1) to (10), wherein the antibody or functional fragment thereof hasantitumor activity;(12) The antibody or functional fragment thereof according to (11),wherein the antibody or functional fragment thereof exhibits antitumoractivity in vivo;(13) The antibody or functional fragment thereof according to any one of(1) to (4), (6), (8), (9), (11) and (12), wherein the antibody consistsof a heavy chain comprising CDRH1 consisting of the amino acid sequencerepresented by SEQ ID NO: 52 (FIG. 60) of the Sequence Listing or anamino acid sequence derived from the amino acid sequence by thesubstitution of one or two amino acids, CDRH2 consisting of the aminoacid sequence represented by SEQ ID NO: 53 (FIG. 61) of the SequenceListing or an amino acid sequence derived from the amino acid sequenceby the substitution of one or two amino acids, and CDRH3 consisting ofthe amino acid sequence represented by SEQ ID NO: 54 (FIG. 62) of theSequence Listing or an amino acid sequence derived from the amino acidsequence by the substitution of one or two amino acids, and a lightchain comprising CDRL1 consisting of the amino acid sequence representedby SEQ ID NO: 61 (FIG. 69) of the Sequence Listing or an amino acidsequence derived from the amino acid sequence by the substitution of oneor two amino acids, CDRL2 consisting of the amino acid sequencerepresented by SEQ ID NO: 62 (FIG. 70) of the Sequence Listing or anamino acid sequence derived from the amino acid sequence by thesubstitution of one or two amino acids, and CDRL3 consisting of theamino acid sequence represented by SEQ ID NO: 63 (FIG. 71) of theSequence Listing or an amino acid sequence derived from the amino acidsequence by the substitution of one or two amino acids, and binds tohuman FGFR2;(14) The antibody or functional fragment thereof according to any one of(1) to (7) and (9) to (12), wherein the antibody consists of a heavychain comprising CDRH1 consisting of the amino acid sequence representedby SEQ ID NO: 55 (FIG. 63) of the Sequence Listing or an amino acidsequence derived from the amino acid sequence by the substitution of oneor two amino acids, CDRH2 consisting of the amino acid sequencerepresented by SEQ ID NO: 56 (FIG. 64) of the Sequence Listing or anamino acid sequence derived from the amino acid sequence by thesubstitution of one or two amino acids, and CDRH3 consisting of theamino acid sequence represented by SEQ ID NO: 57 (FIG. 65) of theSequence Listing or an amino acid sequence derived from the amino acidsequence by the substitution of one or two amino acids, and a lightchain comprising CDRL1 consisting of the amino acid sequence representedby SEQ ID NO: 64 (FIG. 72) of the Sequence Listing or an amino acidsequence derived from the amino acid sequence by the substitution of oneor two amino acids, CDRL2 consisting of the amino acid sequencerepresented by SEQ ID NO: 65 (FIG. 73) of the Sequence Listing or anamino acid sequence derived from the amino acid sequence by thesubstitution of one or two amino acids, and CDRL3 consisting of theamino acid sequence represented by SEQ ID NO: 66 (FIG. 74) of theSequence Listing or an amino acid sequence derived from the amino acidsequence by the substitution of one or two amino acids, and binds tohuman FGFR2;(15) The antibody or functional fragment thereof according to any one of(1) to (7) and (9) to (12), wherein the antibody consists of a heavychain comprising CDRH1 consisting of the amino acid sequence representedby SEQ ID NO: 58 (FIG. 66) of the Sequence Listing or an amino acidsequence derived from the amino acid sequence by the substitution of oneor two amino acids, CDRH2 consisting of the amino acid sequencerepresented by SEQ ID NO: 59 (FIG. 67) of the Sequence Listing or anamino acid sequence derived from the amino acid sequence by thesubstitution of one or two amino acids, and CDRH3 consisting of theamino acid sequence represented by SEQ ID NO: 60 (FIG. 68) of theSequence Listing or an amino acid sequence derived from the amino acidsequence by the substitution of one or two amino acids, and a lightchain comprising CDRL1 consisting of the amino acid sequence representedby SEQ ID NO: 67 (FIG. 75) of the Sequence Listing or an amino acidsequence derived from the amino acid sequence by the substitution of oneor two amino acids, CDRL2 consisting of the amino acid sequencerepresented by SEQ ID NO: 68 (FIG. 76) of the Sequence Listing or anamino acid sequence derived from the amino acid sequence by thesubstitution of one or two amino acids, and CDRL3 consisting of theamino acid sequence represented by SEQ ID NO: 69 (FIG. 77) of theSequence Listing or an amino acid sequence derived from the amino acidsequence by the substitution of one or two amino acids, and binds tohuman FGFR2;(16) The antibody or functional fragment thereof according to (15),wherein the CDRH3 consists of an amino acid sequence derived from theamino acid sequence represented by SEQ ID NO: 60 (FIG. 68) of theSequence Listing by the substitution of one or two amino acids;(17) The antibody or functional fragment thereof according to any one of(1) to (16), wherein the antibody is a monoclonal antibody;(18) The antibody or functional fragment thereof according to any one of(1) to (17), wherein the antibody is a chimeric antibody;(19) The antibody or functional fragment thereof according to any one of(1) to (18), wherein the antibody is a humanized antibody;(20) The antibody or functional fragment thereof according to (19),wherein the antibody is selected from the following (i) to (xix):(i) a humanized antibody (hFR2-14_H19/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 97 (FIG. 105);(ii) a humanized antibody (hFR2-14_H12/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 97 (FIG. 105);(iii) a humanized antibody (hFR2-14_H8/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 89 (FIG. 97);(iv) a humanized antibody (hFR2-14_H11/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 95 (FIG. 103);(v) a humanized antibody (hFR2-14_H5/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 83 (FIG. 91);(vi) a humanized antibody (hFR2-14_H1/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 75 (FIG. 83);(vii) a humanized antibody (hFR2-14_H2/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 77 (FIG. 85);(viii) a humanized antibody (hFR2-14_H3/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 79 (FIG. 87);(ix) a humanized antibody (hFR2-14_H4/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 81 (FIG. 89);(x) a humanized antibody (hFR2-14_H6/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 85 (FIG. 93);(xi) a humanized antibody (hFR2-14_H7/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 87 (FIG. 95);(xii) a humanized antibody (hFR2-14_H9/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 91 (FIG. 99);(xiii) a humanized antibody (hFR2-14_H10/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 93 (FIG. 101);(xiv) a humanized antibody (hFR2-14_H13/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 99 (FIG. 107);(xv) a humanized antibody (hFR2-14_H14/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 101 (FIG. 109);(xvi) a humanized antibody (hFR2-14_H15/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 103 (FIG. 111);(xvii) a humanized antibody (hFR2-14_H16/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 105 (FIG. 113);(xviii) a humanized antibody (hFR2-14_H17/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 107 (FIG. 115); and(xix) a humanized antibody (hFR2-14_H18/L1) comprising a light chaincomprising amino acid positions 21 to 235 of the amino acid sequencerepresented by SEQ ID NO: 73 (FIG. 81), and a heavy chain comprisingamino acid positions 20 to 467 of the amino acid sequence represented bySEQ ID NO: 109 (FIG. 117);(21) The antibody or functional fragment thereof according to any one of(1) to (12), wherein the antibody comprises heavy and light chainscomprising amino acid sequences having 95% or higher identity to theamino acid sequences of the heavy and light chains, respectively, of anantibody according to (20), and binds to human FGFR2; (22) The antibodyor functional fragment thereof according to any one of (1) to (12),wherein the antibody or functional fragment thereof binds to a site onan antigen recognized by an antibody according to any one of (13) to(16) and (20);(23) The antibody or functional fragment thereof according to any one of(1) to (12), wherein the antibody or functional fragment thereofcompetes with an antibody according to any one of (13) to (16) and (20)for binding to human FGFR2;(24) The antibody or functional fragment thereof according to any one of(1) to (12), wherein the antibody or functional fragment thereof bindsto an epitope on human FGFR2, the epitope being constituted by tyrosine(Tyr) at residue 155, threonine (Thr) at residue 157, lysine (Lys) atresidue 176, alanine (Ala) at residue 181, glycine (Gly) at residue 182,glycine (Gly) at residue 183, asparagine (Asn) at residue 184, proline(Pro) at residue 185, methionine (Met) at residue 186, threonine (Thr)at residue 188, glutamine (Gln) at residue 200, glutamic acid (Glu) atresidue 201, glycine (Gly) at residue 205, glycine (Gly) at residue 206,lysine (Lys) at residue 208, valine (Val) at residue 209, arginine (Arg)at residue 210, asparagine (Asn) at residue 211, glutamine (Gin) atresidue 212, histidine (His) at residue 213, tryptophan (Trp) at residue214, and isoleucine (Ile) at residue 217 in the amino acid sequencerepresented by SEQ ID NO: 70 (FIG. 78) or SEQ ID NO: 71 (FIG. 79);(25) The antibody or functional fragment thereof according to any one of(1) to (12), wherein the antibody or functional fragment thereof has aninteraction distance with each of tyrosine (Tyr) at residue 155,threonine (Thr) at residue 157, lysine (Lys) at residue 176, alanine(Ala) at residue 181, glycine (Gly) at residue 182, glycine (Gly) atresidue 183, asparagine (Asn) at residue 184, proline (Pro) at residue185, methionine (Met) at residue 186, threonine (Thr) at residue 188,glutamine (Gin) at residue 200, glutamic acid (Glu) at residue 201,glycine (Gly) at residue 205, glycine (Gly) at residue 206, lysine (Lys)at residue 208, valine (Val) at residue 209, arginine (Arg) at residue210, asparagine (Asn) at residue 211, glutamine (Gin) at residue 212,histidine (His) at residue 213, tryptophan (Trp) at residue 214, andisoleucine (Ile) at residue 217 in the amino acid sequence representedby SEQ ID NO: 70 (FIG. 78) or SEQ ID NO: 71 (FIG. 79);(26) The antibody or functional fragment thereof according to (25),wherein the interaction distance is 6 angstroms or shorter;(27) The antibody or functional fragment thereof according to (25) or(26), wherein the interaction distance is 4 angstroms or shorter;(28) The antibody or functional fragment thereof according to any one of(1) to (12) and (21) to (27), wherein the antibody is a human antibody;(29) The antibody or functional fragment thereof according to any one of(1) to (28), wherein the antibody or functional fragment thereofinhibits the binding of FGF to human FGFR2;(30) The antibody or functional fragment thereof according to any one of(1) to (29), wherein the antibody or functional fragment thereof hasantibody dependent cellular cytotoxic activity and/or antibody dependentcell phagocytosis activity;(31) A nucleotide of any one of the following (i) to (iii):(i) a nucleotide comprising a nucleotide sequence encoding a partial orwhole amino acid sequence of the heavy or light chain of an antibodyaccording to any one of (1) to (30);(ii) a nucleotide consisting of a nucleotide sequence comprising thenucleotide sequence encoding a partial or whole amino acid sequence ofthe heavy or light chain of an antibody according to any one of (1) to(30); and(iii) a nucleotide consisting of the nucleotide sequence encoding apartial or whole amino acid sequence of the heavy or light chain of anantibody according to any one of (1) to (30);(32) A recombinant vector having an insert of a nucleotide according to(31);(33) A recombinant cell comprising a nucleotide according to (31) or arecombinant vector according to (32);(34) A cell producing an antibody or a functional fragment thereofaccording to any one of (1) to (30);(35) A method for producing an antibody or a functional fragment thereofaccording to any one of (1) to (30), comprising the following steps (i)and (ii):(i) culturing a cell according to (33) or (34); and(ii) recovering the antibody or functional fragment thereof according toany one of (1) to (30) from the cultures obtained in the step (i);(36) The antibody or functional fragment thereof according to any one of(1) to (12) which is obtained by a method according to (35);(37) The antibody or functional fragment thereof according to any one of(1) to (30) and (36), wherein 1 to 5 amino acids are deleted from theamino terminus or carboxyl terminus of the heavy or light chain;(38) A modified form of an antibody or a functional fragment thereofaccording to any one of (1) to (30), (36), and (37);(39) The modified form according to (38), wherein a sugar chainmodification is regulated;(40) The modified form according to (39), wherein the antibody isselected from antibodies (i) to (xix) of (20);(41) A pharmaceutical composition comprising an antibody or a functionalfragment thereof according to any one of (1) to (30), (36), and (37), ora modified form according to any one of (38) to (40) as an activeingredient;(42) The pharmaceutical composition according to (41), wherein thepharmaceutical composition is an anticancer agent;(43) The pharmaceutical composition according to (42), wherein thecancer is FGFR2-positive;(44) A composition for testing or diagnosis of cancer comprising anantibody or a functional fragment thereof according to any one of (1) to(30), (36), and (37), or a modified form according to any one of (38) to(40);(45) A composition comprising an antibody or a functional fragmentthereof which has human FGFR2 IIIb selectivity or a modified form of theantibody or the functional fragment;(46) The composition according to (45), wherein the antibody comprises aheavy chain comprising CDRH1 to CDRH3 and a light chain comprising CDRL1to CDRL3 according to (13);(47) The composition according to (46), wherein the antibody comprises aheavy chain variable region having the amino acid sequence representedby SEQ ID NO: 12 (FIG. 20) and a light chain variable region having theamino acid sequence represented by SEQ ID NO: 21 (FIG. 29);(48) The composition according to (46) or (47), wherein the antibody isa chimeric antibody or a rat antibody;(49) The composition according to any one of (45) to (48), wherein thecomposition is for detection or assay of human FGFR2 IIIb;(50) A method for detecting or assaying human FGFR2 IIIb, comprising thestep of contacting a test sample with a composition according to any oneof (45) to (48);(51) A method for detecting or assaying human FGFR2 IIIc, comprising thefollowing steps (i) to (iii):(i) contacting a test sample with a composition comprising an antibodyor a functional fragment thereof which selectively binds to human FGFR2IIIb and human FGFR2 IIIc, or a modified form of the antibody or thefunctional fragment to detect or assay human FGFR2 IIIb and human FGFR2IIIc in the test sample;(ii) contacting the test sample with a composition according to any oneof (45) to (49) to detect or assay the human FGFR2 IIIb in the testsample; and(iii) comparing the results of detection or assay in the step (i) withthe results of detection or assay in the step (ii) or subtracting theresults of detection or assay in the step (ii) from the results ofdetection or assay in the step (i) to obtain detection or assay resultsor a value of the human FGFR2 IIIc in the test sample;(52) The composition according to any one of (44) to (49) or the methodaccording to (50) or (51), wherein the composition or the method is fordiagnosis or testing of a human FGFR2-positive cancer;(53) A method for identifying a recipient individual for apharmaceutical composition according to any one of (41) to (43),comprising the following steps (i) and (ii):(i) contacting an individual-derived sample with a composition accordingto any one of (44) to (49); and(ii) determining the individual to be positive when human FGFR2 isdetected in the sample;(54) The method according to (53), wherein the human FGFR2 is humanFGFR2 IIIb;(55) The method according to (53), wherein the human FGFR2 is humanFGFR2 IIIc and human FGFR2 IIIb;(56) The composition according to any one of (44) to (49), wherein thecomposition is used in a method according to any one of (53) to (55);(57) The method according to any one of (53) to (55) or the compositionaccording to (56), wherein the individual has cancer or is at riskthereof;(58) The pharmaceutical composition according to any one of (41) to(43), wherein the pharmaceutical composition is administered to anindividual identified to be positive by a method according to any one of(53) to (55);(59) A reagent comprising an antibody or a functional fragment thereofaccording to any one of (1) to (30), (36), and (37), or a modified formaccording to any one of (38) to (40);(60) A method for identifying a substance having antitumor activity,comprising the following steps (i) to (iii):(i) contacting a test substance with a protein comprising tyrosine (Tyr)at amino acid position 155 to isoleucine (Ile) at amino acid position217 in the amino acid sequence represented by SEQ ID NO: 70 (FIG. 78) orSEQ ID NO: 71 (FIG. 79);(ii) measuring or determining the distance between the substance andeach of tyrosine (Tyr) at residue 155, threonine (Thr) at residue 157,lysine (Lys) at residue 176, alanine (Ala) at residue 181, glycine (Gly)at residue 182, glycine (Gly) at residue 183, asparagine (Asn) atresidue 184, proline (Pro) at residue 185, methionine (Met) at residue186, threonine (Thr) at residue 188, glutamine (Gin) at residue 200,glutamic acid (Glu) at residue 201, glycine (Gly) at residue 205,glycine (Gly) at residue 206, lysine (Lys) at residue 208, valine (Val)at residue 209, arginine (Arg) at residue 210, asparagine (Asn) atresidue 211, glutamine (Gin) at residue 212, histidine (His) at residue213, tryptophan (Trp) at residue 214, and isoleucine (Ile) at residue217 in the amino acid sequence represented by SEQ ID NO: 70 in theprotein; and(iii) determining the substance to be positive when the substance has aninteraction distance with each of the residues;(61) The method according to (60), further comprising the following step(iv):(iv) assaying the antitumor activity of the substance;(62) The method according to (60) or (61), wherein the substance is anantibody or a functional fragment thereof, or a modified form of theantibody or the functional fragment, or a peptide;(63) A method for producing a substance determined to be positive instep (iii) according to (60) or (61), comprising preparing the substanceby a step including gene recombination, peptide synthesis, or in vitrotranslation;(64) The pharmaceutical composition according to any one of (41) to (43)and (58), further comprising an additional drug;(65) The antibody or functional fragment thereof according to any one of(1) to (30), (36), and (37), or the modified form according to any oneof (38) to (40), wherein the antibody, the functional fragment, or themodified form is conjugated with an additional compound; and(66) The pharmaceutical composition according to any one of (41) to(43), (58), and (64), wherein the pharmaceutical composition comprisesan antibody, a functional fragment, or a modified form according to(65), etc.

Advantageous Effects of Invention

Use of the antibody provided by the present invention enables treatmentor prevention of various cancers and testing or diagnosis of variouscancers.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing results of testing the binding activity ofrat anti-FGFR2 antibodies (FR2-10, FR2-13, and FR2-14) against humanFGFR2 by flow cytometry. The vertical axis represents a relative valueof the average fluorescence intensity assayed by flow cytometry.

FIG. 2 is a diagram showing results of testing for epitopes on humanFGFR2 to which the rat anti-FGFR2 antibodies (FR2-10, FR2-13, andFR2-14) bind by flow cytometry. The vertical axis represents a relativevalue of the average fluorescence intensity assayed by flow cytometry.

FIG. 3A is a diagram showing the signal-neutralizing activity of the ratanti-FGFR2 antibodies (FR2-10, FR2-13, and FR2-14) against human FGFR2IIIb by Elk1 trans-reporter assay.

FIG. 3B is a diagram showing the signal-neutralizing activity of the ratanti-FGFR2 antibodies (FR2-10, FR2-13, and FR2-14) against human FGFR2IIIc by Elk1 trans-reporter assay.

FIG. 4 is a diagram showing the signal inhibitory effect of the ratanti-FGFR2 antibody FR2-10 on FGFR2 by Western blotting. This diagramillustrates that the addition of the rat FR2-10 antibody inhibitedFGFR2, FRS2, and ERK phosphorylation induced by the addition of FGF7 toa human stomach cancer cell line SNU-16.

FIG. 5 is a diagram showing results of testing the binding activity ofhuman chimeric anti-FGFR2 antibodies (cFR2-10, cFR2-13, and cFR2-14)against human FGFR2 by Cell-ELISA.

FIG. 6A is a diagram showing the signal-neutralizing activity of thehuman chimeric anti-FGFR2 antibodies (cFR2-10, cFR2-13, and cFR2-14)against human FGFR2 IIIb by Elk1 trans-reporter assay.

FIG. 6B is a diagram showing the signal-neutralizing activity of thehuman chimeric anti-FGFR2 antibodies (cFR2-10, cFR2-13, and cFR2-14)against human FGFR2 IIIc by Elk1 trans-reporter assay.

FIG. 7 is a diagram showing the ADCC activity of the human chimericanti-FGFR2 antibodies (cFR2-10, cFR2-13, and cFR2-14). 293T-lacZ cellsexpressing human FGFR2 IIIb were used as target cells, and human PBMCwas used as effector cells.

FIG. 8 is a diagram showing the in vivo antitumor activity of the humanchimeric anti-FGFR2 antibodies (cFR2-10, cFR2-13, and cFR2-14) againsthuman stomach cancer cell line SNU-16-transplanted nude mice. FIG. 8A)shows the results for the cFR2-10 antibody. FIG. 8B) shows the resultsfor the cFR2-13 antibody. FIG. 8C) shows the results for the cFR2-14antibody.

FIG. 9 shows the N-terminal amino acid sequence of a band correspondingto the heavy chain of the rat anti-FGFR2 antibody FR2-10 (SEQ ID NO: 1of the Sequence Listing).

FIG. 10 shows the N-terminal amino acid sequence of a band correspondingto the light chain of the rat anti-FGFR2 antibody FR2-10 (SEQ ID NO: 2of the Sequence Listing).

FIG. 11 shows the N-terminal amino acid sequence of a band correspondingto the heavy chain of the rat anti-FGFR2 antibody FR2-13 (SEQ ID NO: 3of the Sequence Listing).

FIG. 12 shows the N-terminal amino acid sequence of a band correspondingto the light chain of the rat anti-FGFR2 antibody FR2-13 (SEQ ID NO: 4of the Sequence Listing).

FIG. 13 shows the N-terminal amino acid sequence of a band correspondingto the heavy chain of the rat anti-FGFR2 antibody FR2-14 (SEQ ID NO: 5of the Sequence Listing).

FIG. 14 shows the N-terminal amino acid sequence of a band correspondingto the light chain of the rat anti-FGFR2 antibody FR2-14 (SEQ ID NO: 6of the Sequence Listing).

FIG. 15 shows a primer for gene amplification of a rat heavy chain (SEQID NO: 7 of the Sequence Listing).

FIG. 16 shows a sequencing primer for the heavy chain of FR2-10 (SEQ IDNO: 8 of the Sequence Listing).

FIG. 17 shows a sequencing primer for the heavy chain of FR2-13 (SEQ IDNO: 9 of the Sequence Listing).

FIG. 18 shows a sequencing primer for the heavy chain of FR2-14 (SEQ IDNO: 10 of the Sequence Listing).

FIG. 19 shows the nucleotide sequence of a cDNA encoding the heavy chainvariable region of the rat anti-FGFR2 antibody FR2-10 (SEQ ID NO: 11 ofthe Sequence Listing).

FIG. 20 shows the amino acid sequence of the heavy chain variable regionof the rat anti-FGFR2 antibody FR2-10 (SEQ ID NO: 12 of the SequenceListing).

FIG. 21 shows the nucleotide sequence of a cDNA encoding the heavy chainvariable region of the rat anti-FGFR2 antibody FR2-13 (SEQ ID NO: 13 ofthe Sequence Listing).

FIG. 22 shows the amino acid sequence of the heavy chain variable regionof the rat anti-FGFR2 antibody FR2-13 (SEQ ID NO: 14 of the SequenceListing).

FIG. 23 shows the nucleotide sequence of a cDNA encoding the heavy chainvariable region of the rat anti-FGFR2 antibody FR2-14 (SEQ ID NO: 15 ofthe Sequence Listing).

FIG. 24 shows the amino acid sequence of the heavy chain variable regionof the rat anti-FGFR2 antibody FR2-14 (SEQ ID NO: 16 of the SequenceListing).

FIG. 25 shows a primer for gene amplification of a rat light chain (SEQID NO: 17 of the Sequence Listing).

FIG. 26 shows a sequencing primer for a rat light chain (SEQ ID NO: 18of the Sequence Listing).

FIG. 27 shows a sequencing primer for the light chain of FR2-10 (SEQ IDNO: 19 of the Sequence Listing).

FIG. 28 shows the nucleotide sequence of a cDNA encoding the light chainvariable region of the rat anti-FGFR2 antibody FR2-10 (SEQ ID NO: 20 ofthe Sequence Listing).

FIG. 29 shows the amino acid sequence of the light chain variable regionof the rat anti-FGFR2 antibody FR2-10 (SEQ ID NO: 21 of the SequenceListing).

FIG. 30 shows a primer for gene amplification of the rat FR2-13 orFR2-14 light chain (SEQ ID NO: 22 of the Sequence Listing).

FIG. 31 shows the nucleotide sequence of a cDNA encoding the light chainvariable region of the rat anti-FGFR2 antibody FR2-13 (SEQ ID NO: 23 ofthe Sequence Listing).

FIG. 32 shows the amino acid sequence of the light chain variable regionof the rat anti-FGFR2 antibody FR2-13 (SEQ ID NO: 24 of the SequenceListing).

FIG. 33 shows the nucleotide sequence of a cDNA encoding the light chainvariable region of the rat anti-FGFR2 antibody FR2-14 (SEQ ID NO: 25 ofthe Sequence Listing).

FIG. 34 shows the amino acid sequence of the light chain variable regionof the rat anti-FGFR2 antibody FR2-14 (SEQ ID NO: 26 of the SequenceListing).

FIG. 35 shows a DNA fragment comprising a DNA sequence encoding theamino acids of a human κ chain secretory signal sequence and a human κchain constant region (SEQ ID NO: 27 of the Sequence Listing).

FIG. 36 shows a primer F for a light chain expression vector (SEQ ID NO:28 of the Sequence Listing).

FIG. 37 shows a primer R for a light chain expression vector (SEQ ID NO:29 of the Sequence Listing).

FIG. 38 shows a DNA fragment comprising a DNA sequence encoding theamino acids of a human heavy chain signal sequence and a human IgG1constant region (SEQ ID NO: 30 of the Sequence Listing).

FIG. 39 shows the nucleotide sequence of the light chain of humanchimeric FR2-10 (cFR2-10) (SEQ ID NO: 31 of the Sequence Listing). Inthis sequence, nucleotide positions 1 to 60 represent a signal sequence,which is usually not contained in the nucleotide sequences of most ofmature cFR2-10 light chains.

FIG. 40 shows the amino acid sequence of the light chain of humanchimeric FR2-10 (cFR2-10) (SEQ ID NO: 32 of the Sequence Listing). Inthis sequence, amino acid positions 1 to 20 represent a signal sequence,which is usually not contained in the amino acid sequences of most ofmature cFR2-10 light chains.

FIG. 41 shows a primer set F for the light chain of human chimericFR2-10 (SEQ ID NO: 33 of the Sequence Listing).

FIG. 42 shows a primer set R for the light chain of human chimericFR2-10 (SEQ ID NO: 34 of the Sequence Listing).

FIG. 43 shows the nucleotide sequence of the heavy chain of humanchimeric FR2-10 (cFR2-10) (SEQ ID NO: 35 of the Sequence Listing). Inthis sequence, nucleotide positions 1 to 57 represent a signal sequence,which is usually not contained in the nucleotide sequences of most ofmature cFR2-10 heavy chains.

FIG. 44 shows the amino acid sequence of the heavy chain of humanchimeric FR2-10 (cFR2-10) (SEQ ID NO: 36 of the Sequence Listing). Inthis sequence, amino acid positions 1 to 19 represent a signal sequence,which is usually not included in the amino acid sequence of most ofmature cFR2-10 heavy chains.

FIG. 45 shows a primer set F for the heavy chain of human chimericFR2-10 (SEQ ID NO: 37 of the Sequence Listing).

FIG. 46 shows a primer set R for the heavy chain of human chimericFR2-10 (SEQ ID NO: 38 of the Sequence Listing).

FIG. 47 shows the nucleotide sequence of the light chain of humanchimeric FR2-13 (cFR2-13) (SEQ ID NO: 39 of the Sequence Listing). Inthis sequence, nucleotide positions 1 to 60 represent a signal sequence,which is usually not contained in the nucleotide sequences of most ofmature cFR2-13 light chains.

FIG. 48 shows the amino acid sequence of the light chain of humanchimeric FR2-13 (cFR2-13) (SEQ ID NO: 40 of the Sequence Listing). Inthis sequence, amino acid positions 1 to 20 represent a signal sequence,which is usually not contained in the amino acid sequences of most ofmature cFR2-13 light chains.

FIG. 49 shows a primer F for the light chain of human chimeric FR2-13(SEQ ID NO: 41 of the Sequence Listing).

FIG. 50 shows a primer R for the light chain of human chimeric FR2-13(SEQ ID NO: 42 of the Sequence Listing).

FIG. 51 shows the nucleotide sequence of the heavy chain of humanchimeric FR2-13 (cFR2-13) (SEQ ID NO: 43 of the Sequence Listing). Inthis sequence, nucleotide positions 1 to 57 represent a signal sequence,which is usually not contained in the nucleotide sequences of most ofmature cFR2-13 heavy chains.

FIG. 52 shows the amino acid sequence of the heavy chain of humanchimeric FR2-13 (cFR2-13) (SEQ ID NO: 44 of the Sequence Listing). Inthis sequence, amino acid positions 1 to 19 represent a signal sequence,which is usually not contained in the amino acid sequences of most ofmature cFR2-13 heavy chains.

FIG. 53 shows a primer F for the heavy chain of human chimeric FR2-13(SEQ ID NO: 45 of the Sequence Listing).

FIG. 54 shows a primer R for the heavy chain of human chimeric FR2-13(SEQ ID NO: 46 of the Sequence Listing).

FIG. 55 shows the nucleotide sequence of the light chain of humanchimeric FR2-14 (cFR2-14) (SEQ ID NO: 47 of the Sequence Listing). Inthis sequence, nucleotide positions 1 to 60 represent a signal sequence,which is usually not contained in the nucleotide sequences of most ofmature cFR2-14 light chains.

FIG. 56 shows the amino acid sequence of the light chain of humanchimeric FR2-14 (cFR2-14) (SEQ ID NO: 48 of the Sequence Listing). Inthis sequence, amino acid positions 1 to 20 represent a signal sequence,which is usually not contained in the amino acid sequences of most ofmature cFR2-14 light chains.

FIG. 57 shows a primer for the light chain of human chimeric FR2-14 (SEQID NO: 49 of the Sequence Listing).

FIG. 58 shows the nucleotide sequence of the heavy chain of humanchimeric FR2-14 (cFR2-14) (SEQ ID NO: 50 of the Sequence Listing). Inthis sequence, nucleotide positions 1 to 57 represent a signal sequence,which is usually not contained in the nucleotide sequences of most ofmature cFR2-14 heavy chains.

FIG. 59 shows the amino acid sequence of the heavy chain of humanchimeric FR2-14 (cFR2-14) (SEQ ID NO: 51 of the Sequence Listing). Inthis sequence, amino acid positions 1 to 19 represent a signal sequence,which is usually not contained in the amino acid sequences of most ofmature cFR2-14 heavy chains.

FIG. 60 shows the amino acid sequence of the heavy chain CDR1 of the ratanti-FGFR2 antibody FR2-10 (SEQ ID NO: 52 of the Sequence Listing).

FIG. 61 shows the amino acid sequence of the heavy chain CDR2 of the ratanti-FGFR2 antibody FR2-10 (SEQ ID NO: 53 of the Sequence Listing).

FIG. 62 shows the amino acid sequence of the heavy chain CDR3 of the ratanti-FGFR2 antibody FR2-10 (SEQ ID NO: 54 of the Sequence Listing).

FIG. 63 shows the amino acid sequence of the heavy chain CDR1 of the ratanti-FGFR2 antibody FR2-13 (SEQ ID NO: 55 of the Sequence Listing).

FIG. 64 shows the amino acid sequence of the heavy chain CDR2 of the ratanti-FGFR2 antibody FR2-13 (SEQ ID NO: 56 of the Sequence Listing).

FIG. 65 shows the amino acid sequence of the heavy chain CDR3 of the ratanti-FGFR2 antibody FR2-13 (SEQ ID NO: 57 of the Sequence Listing).

FIG. 66 shows the amino acid sequence of the heavy chain CDR1 of the ratanti-FGFR2 antibody FR2-14 (SEQ ID NO: 58 of the Sequence Listing).

FIG. 67 shows the amino acid sequence of the heavy chain CDR2 of the ratanti-FGFR2 antibody FR2-14 (SEQ ID NO: 59 of the Sequence Listing).

FIG. 68 shows the amino acid sequence of the heavy chain CDR3 of the ratanti-FGFR2 antibody FR2-14 (SEQ ID NO: 60 of the Sequence Listing).

FIG. 69 shows the amino acid sequence of the light chain CDR1 of the ratanti-FGFR2 antibody FR2-10 (SEQ ID NO: 61 of the Sequence Listing).

FIG. 70 shows the amino acid sequence of the light chain CDR2 of the ratanti-FGFR2 antibody FR2-10 (SEQ ID NO: 62 of the Sequence Listing).

FIG. 71 shows the amino acid sequence of the light chain CDR3 of the ratanti-FGFR2 antibody FR2-10 (SEQ ID NO: 63 of the Sequence Listing).

FIG. 72 shows the amino acid sequence of the light chain CDR1 of the ratanti-FGFR2 antibody FR2-13 (SEQ ID NO: 64 of the Sequence Listing).

FIG. 73 shows the amino acid sequence of the light chain CDR2 of the ratanti-FGFR2 antibody FR2-13 (SEQ ID NO: 65 of the Sequence Listing).

FIG. 74 shows the amino acid sequence of the light chain CDR3 of the ratanti-FGFR2 antibody FR2-13 (SEQ ID NO: 66 of the Sequence Listing).

FIG. 75 shows the amino acid sequence of the light chain CDR1 of the ratanti-FGFR2 antibody FR2-14 (SEQ ID NO: 67 of the Sequence Listing).

FIG. 76 shows the amino acid sequence of the light chain CDR2 of the ratanti-FGFR2 antibody FR2-14 (SEQ ID NO: 68 of the Sequence Listing).

FIG. 77 shows the amino acid sequence of the light chain CDR3 of the ratanti-FGFR2 antibody FR2-14 (SEQ ID NO: 69 of the Sequence Listing).

FIG. 78 shows the amino acid sequence of human FGFR2 IIIb (SEQ ID NO: 70of the Sequence Listing).

FIG. 79 shows the amino acid sequence of human FGFR2 IIIc (SEQ ID NO: 71of the Sequence Listing).

FIG. 80 shows the nucleotide sequence of hFR2-14_L1 (SEQ ID NO: 72 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 60represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature light chains hFR2-14_L1.

FIG. 81 shows the amino acid sequence of hFR2-14_L1 (SEQ ID NO: 73 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 20represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature light chains hFR2-14_L1.

FIG. 82 shows the nucleotide sequence of hFR2-14_H1 (SEQ ID NO: 74 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H1.

FIG. 83 shows the amino acid sequence of hFR2-14_H1 (SEQ ID NO: 75 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H1.

FIG. 84 shows the nucleotide sequence of hFR2-14_H2 (SEQ ID NO: 76 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H2.

FIG. 85 shows the amino acid sequence of hFR2-14_H2 (SEQ ID NO: 77 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H2.

FIG. 86 shows the nucleotide sequence of hFR2-14_H3 (SEQ ID NO: 78 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H3.

FIG. 87 shows the amino acid sequence of hFR2-14_H3 (SEQ ID NO: 79 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H3.

FIG. 88 shows the nucleotide sequence of hFR2-14_H4 (SEQ ID NO: 80 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H4.

FIG. 89 shows the amino acid sequence of hFR2-14_H4 (SEQ ID NO: 81 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H4.

FIG. 90 shows the nucleotide sequence of hFR2-14_H5 (SEQ ID NO: 82 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H5.

FIG. 91 shows the amino acid sequence of hFR2-14_H5 (SEQ ID NO: 83 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H5.

FIG. 92 shows the nucleotide sequence of hFR2-14_H6 (SEQ ID NO: 84 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H6.

FIG. 93 shows the amino acid sequence of hFR2-14_H6 (SEQ ID NO: 85 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H6.

FIG. 94 shows the nucleotide sequence of hFR2-14_H7 (SEQ ID NO: 86 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H7.

FIG. 95 shows the amino acid sequence of hFR2-14_H7 (SEQ ID NO: 87 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H7.

FIG. 96 shows the nucleotide sequence of hFR2-14_H8 (SEQ ID NO: 88 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H8.

FIG. 97 shows the amino acid sequence of hFR2-14_H8 (SEQ ID NO: 89 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H8.

FIG. 98 shows the nucleotide sequence of hFR2-14_H9 (SEQ ID NO: 90 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H9.

FIG. 99 shows the amino acid sequence of hFR2-14_H9 (SEQ ID NO: 91 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H9.

FIG. 100 shows the nucleotide sequence of hFR2-14_H10 (SEQ ID NO: 92 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H10.

FIG. 101 shows the amino acid sequence of hFR2-14_H10 (SEQ ID NO: 93 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H10.

FIG. 102 shows the nucleotide sequence of hFR2-14_H11 (SEQ ID NO: 94 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H11.

FIG. 103 shows the amino acid sequence of hFR2-14_H11 (SEQ ID NO: 95 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H11.

FIG. 104 shows the nucleotide sequence of hFR2-14_H12 or hFR2-14_H19(SEQ ID NO: 96 of the Sequence Listing). In this sequence, nucleotidepositions 1 to 57 represent a signal sequence, which is usually notincluded in the nucleotide sequence of most of mature heavy chainshFR2-14_H12 or hFR2-14_H19.

FIG. 105 shows the amino acid sequence of hFR2-14_H12 or hFR2-14_H19(SEQ ID NO: 97 of the Sequence Listing). In this sequence, amino acidpositions 1 to 19 represent a signal sequence, which is usually notincluded in the amino acid sequence of most of mature heavy chainshFR2-14_H12 or hFR2-14_H19.

FIG. 106 shows the nucleotide sequence of hFR2-14_H13 (SEQ ID NO: 98 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H13.

FIG. 107 shows the amino acid sequence of hFR2-14_H13 (SEQ ID NO: 99 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H13.

FIG. 108 shows the nucleotide sequence of hFR2-14_H14 (SEQ ID NO: 100 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H14.

FIG. 109 shows the amino acid sequence of hFR2-14_H14 (SEQ ID NO: 101 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H14.

FIG. 110 shows the nucleotide sequence of hFR2-14_H15 (SEQ ID NO: 102 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H15.

FIG. 111 shows the amino acid sequence of hFR2-14_H15 (SEQ ID NO: 103 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H15.

FIG. 112 shows the nucleotide sequence of hFR2-14_H16 (SEQ ID NO: 104 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H16.

FIG. 113 shows the amino acid sequence of hFR2-14_H16 (SEQ ID NO: 105 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H16.

FIG. 114 shows the nucleotide sequence of hFR2-14_H17 (SEQ ID NO: 106 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H17.

FIG. 115 shows the amino acid sequence of hFR2-14_H17 (SEQ ID NO: 107 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H17.

FIG. 116 shows the nucleotide sequence of hFR2-14_H18 (SEQ ID NO: 108 ofthe Sequence Listing). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not included in thenucleotide sequence of most of mature heavy chains hFR2-14_H18.

FIG. 117 shows the amino acid sequence of hFR2-14_H18 (SEQ ID NO: 109 ofthe Sequence Listing). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not included in the aminoacid sequence of most of mature heavy chains hFR2-14_H18.

FIG. 118 shows a primer VH3A-F for an hFR2-14_H2 type heavy chain (SEQID NO: 110 of the Sequence Listing).

FIG. 119 shows a primer VH3A-R for an hFR2-14_H2 type heavy chain (SEQID NO: 111 of the Sequence Listing).

FIG. 120 shows a primer D23fw for gene amplification of D2 (SEQ ID NO:112 of the Sequence Listing).

FIG. 121 shows a primer D23rv for gene amplification of D2 (SEQ ID NO:113 of the Sequence Listing).

FIG. 122 is a diagram showing results of assaying the binding activityof 4 types of humanized anti-FGFR2 antibodies (hFR2-14_H1/L1 tohFR2-14_H4/L1) and the human chimeric anti-FGFR2 antibody (cFR2-14)against each human FGFR2 variant protein using Biacore. Each antibodywas expressed in 293F cells and purified for use in the assay.

FIG. 123 is a diagram showing results of assaying the binding activityof 15 types of humanized anti-FGFR2 antibodies (hFR2-14_H3/L1 andhFR2-14_H5/L1 to hFR2-14_H18/L1) against a human FGFR2 IIIc variantprotein using Biacore. Each antibody was expressed in 293F cells, andits culture supernatant was used in the assay.

FIG. 124 is a diagram showing results of testing the humanFGFR2-selective binding activity of 3 types of humanized anti-FGFR2antibodies (hFR2-14_H3/L1, hFR2-14_H8/L1, and hFR2-14_H12/L1) byCell-ELISA.

FIG. 125A is a diagram showing the thermograms of 5 types of humanizedanti-FGFR2 antibodies.

FIG. 125B is a diagram showing the thermograms of 5 types of humanizedanti-FGFR2 antibodies.

FIG. 125C is a diagram showing the Tm values of 10 types of humanizedanti-FGFR2 antibodies.

FIG. 126 is a diagram showing the KD values of hFR2-14_H1/L1,hFR2-14_H2/L1, hFR2-14_H3/L1, hFR2-14_H4/L1, hFR2-14_H5/L1,hFR2-14_H8/L1, hFR2-14_H9/L1, hFR2-14_H11/L1, hFR2-14_H12/L1, andhFR2-14_H19/L1 antibody analytes for an antigen before and afterdegradation.

FIG. 127A is a diagram showing the signal-neutralizing activity of thehumanized anti-FGFR2 antibodies (hFR2-14_H1/L1, hFR2-14_H2/L1,hFR2-14_H3/L1, and hFR2-14_H4/L1) and the human chimeric anti-FGFR2antibody (cFR2-14) against human FGFR2 IIIb by Elk1 trans-reporterassay.

FIG. 127B is a diagram showing the signal-neutralizing activity of thehumanized anti-FGFR2 antibodies (hFR2-14_H1/L1, hFR2-14_H2/L1,hFR2-14_H3/L1, and hFR2-14_H4/L1) and the human chimeric anti-FGFR2antibody (cFR2-14) against human FGFR2 IIIc by Elk1 trans-reporterassay.

FIG. 128A is a diagram showing the signal-neutralizing activity of thehumanized anti-FGFR2 antibodies (hFR2-14_H3/L1, hFR2-14_H5/L1,hFR2-14_H6/L1, hFR2-14_H7/L1, and hFR2-14_H8/L1) against human FGFR2IIIb by Elk1 trans-reporter assay.

FIG. 128B is a diagram showing the signal-neutralizing activity of thehumanized anti-FGFR2 antibodies (hFR2-14_H9/L1, hFR2-14_H10/L1,hFR2-14_H11/L1, hFR2-14_H12/L1, and hFR2-14_H13/L1) against human FGFR2IIIb by Elk1 trans-reporter assay.

FIG. 128C is a diagram showing the signal-neutralizing activity of thehumanized anti-FGFR2 antibodies (hFR2-14_H14/L1, hFR2-14_H15/L1,hFR2-14_H16/L1, hFR2-14_H17/L1, and hFR2-14_H18/L1) against human FGFR2IIIb by Elk1 trans-reporter assay.

FIG. 129A is a diagram showing the signal-neutralizing activity of thehumanized anti-FGFR2 antibodies (hFR2-14_H12/L1 and hFR2-14_H19/L1)against human FGFR2 IIIb by Elk1 trans-reporter assay.

FIG. 129B is a diagram showing the signal-neutralizing activity of thehumanized anti-FGFR2 antibodies (hFR2-14_H12/L1 and hFR2-14_H19/L1)against human FGFR2 IIIc by Elk1 trans-reporter assay.

FIG. 130A is a diagram showing the ADCC activity of the humanizedanti-FGFR2 antibodies (hFR2-14_H1/L1 and hFR2-14_H2/L1). 293T-lacZ cellsexpressing human FGFR2 IIIb were used as target cells, and human PBMCwas used as effector cells.

FIG. 130B is a diagram showing the ADCC activity of the humanizedanti-FGFR2 antibodies (hFR2-14_H3/L1 and hFR2-14_H4/L1) and the humanchimeric anti-FGFR2 antibody (cFR2-14). 293T-lacZ cells expressing humanFGFR2 IIIb were used as target cells, and human PBMC was used aseffector cells.

FIG. 131 is a diagram showing the ADCC activity of the humanizedanti-FGFR2 antibodies (hFR2-14_H3/L1, hFR2-14_H8/L1, andhFR2-14_H12/L1). NCI-H716 cells expressing human FGFR2 were used astarget cells, and human PBMC was used as effector cells.

FIG. 132 is a diagram showing the ADCC activity of the humanizedanti-FGFR2 antibodies (hFR2-14_H12/L1 and hFR2-14_H19/L1). NCI-H716cells (FIG. 132A)), SNU-16 cells (FIG. 132B)), or KATO III cells (FIG.132C)) expressing human FGFR2 were used as target cells, and human PBMCwas used as effector cells.

FIG. 133 is a diagram showing the ADCP activity of the humanizedanti-FGFR2 antibodies (hFR2-14_H12/L1 and hFR2-14_H19/L1). NCI-H716cells (FIG. 133A)) or KATO III cells (FIG. 133B)) expressing human FGFR2were used as target cells, and macrophage-like cells differentiated fromhuman PBMC were used as effector cells.

FIG. 134A is a diagram showing the in vivo antitumor activity of thehumanized anti-FGFR2 antibody hFR2-14_H1/L1 against human stomach cancercell line SNU-16-transplanted nude mice.

FIG. 134B is a diagram showing the in vivo antitumor activity of thehumanized anti-FGFR2 antibody hFR2-14_H2/L1 against human stomach cancercell line SNU-16-transplanted nude mice.

FIG. 134C is a diagram showing the in vivo antitumor activity of thehumanized anti-FGFR2 antibody hFR2-14_H3/L1 against human stomach cancercell line SNU-16-transplanted nude mice.

FIG. 134D is a diagram showing the in vivo antitumor activity of thehumanized anti-FGFR2 antibody hFR2-14_H4/L1 against human stomach cancercell line SNU-16-transplanted nude mice.

FIG. 135A is a diagram showing the in vivo antitumor activity of thehumanized anti-FGFR2 antibody hFR2-14_H5/L1 against human stomach cancercell line SNU-16-subcutaneously transplanted models.

FIG. 135B is a diagram showing the in vivo antitumor activity of thehumanized anti-FGFR2 antibody hFR2-14_H8/L1 against human stomach cancercell line SNU-16-subcutaneously transplanted models.

FIG. 135C is a diagram showing the in vivo antitumor activity of thehumanized anti-FGFR2 antibody hFR2-14_H9/L1 against human stomach cancercell line SNU-16-subcutaneously transplanted models.

FIG. 135D is a diagram showing the in vivo antitumor activity of thehumanized anti-FGFR2 antibody hFR2-14_H11/L1 against human stomachcancer cell line SNU-16-subcutaneously transplanted models.

FIG. 135E is a diagram showing the in vivo antitumor activity of thehumanized anti-FGFR2 antibody hFR2-14_H12/L1 against human stomachcancer cell line SNU-16-subcutaneously transplanted models.

FIG. 135F is a diagram showing the in vivo antitumor activity of thehumanized anti-FGFR2 antibody hFR2-14_H19/L1 against human stomachcancer cell line SNU-16-subcutaneously transplanted models.

FIG. 136 is a diagram showing the in vivo antitumor activity of thehumanized anti-FGFR2 antibodies (hFR2-14_H12/L1 and hFR2-14_H19/L1)against human colorectal cancer cell line NCI-H716 tumor block models.FIG. 136(A) shows the results for hFR2-14_H12/L1. FIG. 136(B) shows theresults for hFR2-14_H19/L1.

FIG. 137 is a diagram showing the in vivo antitumor activity of thehumanized anti-FGFR2 antibodies (hFR2-14_H12/L1 and hFR2-14_H19/L1)against human colorectal cancer cell line NCI-H716-luc-peritoneallydisseminated models. FIG. 137(A) shows the results showing luciferaseactivity. FIG. 137(B) shows the results showing survival rates.

FIG. 138 is a diagram showing a ribbon model of an FGFR2D2/H3L1Fabcomplex. FGFR2D2 is shown at the lower left. The H chain (dark gray) ofH3L1Fab is shown from the center to the upper right. The L chain ofH3L1Fab is shown at the right thereof.

FIG. 139 is a diagram showing the superposition of FGFR2/FGF1 andFGFR2D2/H3L1Fab. FGFRD3 (IgD3) is shown at the lower center. FGF1 (darkgray) is shown above FGFRD3. FGFRD2 is shown above FGF1. The H chain(dark gray) and L chain of H3L1Fab are each shown at the upper right ofFGFRD2.

FIG. 140 is a diagram showing results of immunostainingblocks of 293αcells forced to express each molecule of the FGFR family, using the ratantibody FR2-10.

FIG. 141 is a diagram showing results of immunostaining blocks of SNU-16cells (FIGS. 141A and 141D), KATO III cells (FIGS. 141B and 141E), andNCI-H716 cells (FIGS. 141C and 141F). FIGS. 141A to 141C show theresults obtained using the rat antibody FR2-10. FIGS. 141D to 141F showthe results obtained using a commercially available antibody.

FIG. 142 is a diagram showing results of immunostaining xenograft tumorsamples of SNU-16 cells (FIG. 142A), KATO III cells (FIG. 142B), andNCI-H716 cells (FIG. 142C) using the rat antibody FR2-10.

FIG. 143 is a diagram showing the activity of inhibiting the binding ofa ligand FGF7 to its receptor FGFR2 by the cFR2-10, hFR2-14_H12/L1, andhFR2-14_H19/L1 antibodies.

DESCRIPTION OF EMBODIMENTS 1. Definitions

In the present invention, the term “gene” means a nucleotide comprisinga nucleotide sequence encoding the amino acids of a protein, or itscomplementary strand. The “gene” is meant to include, for example, apolynucleotide, an oligonucleotide, DNA, mRNA, cDNA, and cRNA as thenucleotide comprising a nucleotide sequence encoding the amino acids ofa protein, or its complementary strand. Such a gene is asingle-stranded, double-stranded, or triple or more stranded nucleotide.The “gene” is also meant to include an association of DNA and RNAstrands, a mixture of ribonucleotides (RNAs) and deoxyribonucleotides(DNAs) on one nucleotide strand, and a double-stranded or triple or morestranded nucleotide comprising such a nucleotide strand. Examples of the“FGFR2 gene” of the present invention can include DNA, mRNA, cDNA, andcRNA comprising a nucleotide sequence encoding the amino acid sequenceof the FGFR2 protein.

In the present invention, the term “nucleotide” has the same meaning asa “nucleic acid” and is also meant to include, for example, DNA, RNA, aprobe, an oligonucleotide, a polynucleotide, and a primer. Such anucleotide is a single-stranded, double-stranded, or triple or morestranded nucleotide. The “nucleotide” is also meant to include anassociation of DNA and RNA strands, a mixture of ribonucleotides (RNAs)and deoxyribonucleotides (DNAs) on one nucleotide strand, and anassociate of two strands or three or more strands comprising such anucleotide strand.

In the present invention, the terms “polypeptide”, “peptide”, and“protein” have the same meaning.

In the present invention, the term “antigen” has the same meaning as“immunogen”.

In the present invention, the term “cell” also includes, for example,various cells derived from individual animals, subcultured cells,primary cultured cells, cell lines, recombinant cells, and microbialcells.

In the present invention, antibodies recognizing FGFR2, FGFR2 IIIb,FGFR2 IIIc, FGFR3, FGFRs, and the like are also referred to as an“anti-FGFR2 antibody”, an “anti-FGFR2 IIIb antibody”, an “anti-FGFR2IIIc antibody”, an “anti-FGFR3 antibody”, and an “anti-FGFRs antibody”,respectively. These antibodies include chimeric antibodies, humanizedantibodies, human antibodies, and the like.

In the present invention, the term “functional fragment of the antibody”means an antibody fragment that exerts at least a portion of functionsexerted by the original antibody. Examples of the “functional fragmentof the antibody” can include, but are not limited to, Fab, F(ab′)2,scFv, Fab′, and single chain immunoglobulin. Such a functional fragmentof the antibody may be obtained by treating a full-length molecule ofthe antibody protein with an enzyme such as papain or pepsin or may be arecombinant protein produced in an appropriate host cell using arecombinant gene.

In the present invention, the “site” to which an antibody binds, i.e.,the “site” recognized by an antibody, means a partial peptide or partialconformation on an antigen bound or recognized by the antibody. In thepresent invention, such a site is also referred to as an epitope or anantibody binding site. Examples of the site on the FGFR2 protein boundor recognized by the anti-FGFR2 antibody of the present invention caninclude a partial peptide or partial conformation on the FGFR2 protein.

The heavy and light chains of an antibody molecule are known to eachhave three complementarity determining regions (CDRs). Thecomplementarity determining regions are also called hypervariabledomains. These regions are located in the variable regions of theantibody heavy and light chains. These sites have a particularly highlyvariable primary structure and are usually separated at three positionson the respective primary structures of heavy and light chainpolypeptide strands. In the present invention, the complementaritydetermining regions of the antibody are referred to as CDRH1, CDRH2, andCDRH3 from the amino terminus of the heavy chain amino acid sequence forthe complementarity determining regions of the heavy chain and as CDRL1,CDRL2, and CDRL3 from the amino terminus of the light chain amino acidsequence for the complementarity determining regions of the light chain.These sites are proximal to each other on the three-dimensionalstructure and determine specificity for the antigen to be bound.

In the present invention, the term “antibody mutant” means a polypeptidethat has an amino acid sequence derived from the amino acid sequence ofthe original antibody by the substitution, deletion, addition, and/orinsertion (hereinafter, collectively referred to as a “mutation”) ofamino acid(s) and binds to the FGFR2 protein of the present invention.The number of mutated amino acids in such an antibody mutant is 1 to 2,1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to12, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 40, or 1 to 50. Such anantibody mutant is also encompassed by the “antibody” of the presentinvention.

In the present invention, the term “several” in “1 to several” refers to3 to 10.

Examples of activities or properties exerted by the antibody of thepresent invention can include biological activities or physicochemicalproperties and can specifically include various biological activities,binding activity against an antigen or an epitope, stability duringproduction or storage, and thermal stability.

In the present invention, the phrase “hybridizing under stringentconditions” means hybridization under conditions involving hybridizationat 65° C. in a solution containing 5×SSC, followed by washing at 65° C.for 20 minutes in an aqueous solution containing 2×SSC-0.1% SDS, at 65°C. for 20 minutes in an aqueous solution containing 0.5×SSC-0.1% SDS,and at 65° C. for 20 minutes in an aqueous solution containing0.2×SSC-0.1% SDS, or hybridization under conditions equivalent thereto.SSC means an aqueous solution of 150 mM NaCl-15 mM sodium citrate, andn×SSC means SSC with an n-fold concentration.

In the present invention, the term “cytotoxicity” refers to somepathological change brought about to cells and means not only directtrauma but every structural or functional damage to cells, including DNAcleavage, formation of base dimers, chromosomal break, damage on mitoticapparatus, and reduction in the activities of various enzymes.

In the present invention, the term “cytotoxic activity” means activitythat causes the cytotoxicity mentioned above.

In the present invention, the term “antibody dependent cellularcytotoxic activity”, also called “ADCC activity”, means the effect oractivity of damaging target cells such as tumor cells by NK cells viaantibodies.

In the present invention, the term “antibody dependent cell phagocytosisactivity”, also called “ADCP activity”, means the effect or activity ofenglobing target cells such as tumor cells by monocyte or macrophagecells via antibodies. This activity is also referred to as “antibodydependent phagocytic effect or activity”.

In the present invention, the term “complement dependent cytotoxicactivity”, also called “CDC activity”, means the effect or activity ofdamaging target cells such as tumor cells by complement via antibodies.

In the present invention, the term “cancer” has the same meaning as“tumor”.

In the present invention, the term “immunohistochemistry (IHC)” means ahistological (histochemical) approach of detecting an antigen in atissue preparation. The immunohistochemistry is synonymous with an“immune antibody method” and has the same meaning as “immunostaining”.

2. Antigenic Protein

(2-1) Properties

FGFRs are receptor proteins that bind to fibroblast growth factors(FGFs). In the present invention, FGFRs are derived from vertebrates,preferably mammals, more preferably humans. Human FGFs and FGFRs areclassified into 22 FGFs (FGF1 to FGF14 and FGF16 to FGF23) and 4 FGFRs(FGFR1 to FGFR4) having a tyrosine kinase domain, respectively. TheseFGFRs are each composed of an extracellular region comprising a ligandbinding site composed of 2 or 3 immunoglobulin-like domains (IgD1 toIgD3), a single-pass transmembrane region, and an intracellular regioncomprising the tyrosine kinase domain. Of them, FGFR1, FGFR2, and FGFR3each have two splicing variants called IIIb and IIIc. These isoformsdiffer in the sequence of approximately 50 amino acids in the latterhalf of IgD3 and exhibit distinctive tissue distribution and ligandspecificity. FGFRs have the following activities: (1) binding to FGFs;(2) this binding dimerizes the FGFRs; (3) this dimerizationphosphorylates the FGFRs at their particular tyrosine residues; (4) thisphosphorylation promotes the recruitment of adaptor proteins such asFGFR substrate 2a (FRS2a); and (5) this transduces signals generated byFGF stimulation to cells or tissues expressing the FGFRs or activatessignal transduction.

The FGFR2 protein according to the present invention has the followingproperties:

(i) Binding to FGF.

The FGFR2 IIIb protein typically binds to one or two or more FGFsselected from the group consisting of FGF1, FGF3, FGF7 (KGF), FGF10,FGF22, and FGF23. The FGFR2 IIIb protein may bind to other FGFs and maynot bind to mutated forms of the FGFs included in the above group.

The FGFR2 IIIc protein typically binds to one or two or more FGFsselected from the group consisting of FGF1, FGF2, FGF4, FGF6, FGF9,FGF17, FGF18, FGF21, and FGF23. The FGFR2 IIIc protein may bind to otherFGFs and may not bind to mutated forms of the FGFs included in the abovegroup.

(ii) Transducing signals generated by FGF stimulation intoFGFR2-expressing cells or tissues

Examples of the transduction of signals generated by FGF stimulation caninclude, but are not particularly limited to, FGFR2 autophosphorylation,recruitment of FGFR substrates and promotion thereof, and activation ofsignaling pathways such as MAPK, PI3K, Akt, and extracellularsignal-regulated kinase (ERK) pathways via these events. Examples of theFGFR substrates can include FGFR substrate 2α (FRS2α).

Testing methods for evaluating the activation of this signaltransduction and the inhibition thereof are not particularly limited andcan be arbitrarily selected from methods known in the art. Examplesthereof can include evaluation systems for ERK signal transduction, andElk1 luciferase reporter assay described later.

(iii) The FGFR2 IIIb protein according to the present inventioncomprises an amino acid sequence described in any one of the following(a) to (d) (hereinafter, referred to as an “FGFR2 IIIb amino acidsequence”), consists of an amino acid sequence comprising the FGFR2 IIIbamino acid sequence, or consists of the FGFR2 IIIb amino acid sequence:

(a) the amino acid sequence represented by SEQ ID NO: 70 (FIG. 78) ofthe Sequence Listing;(b) an amino acid sequence that exhibits 80% or higher, 82% or higher,84% or higher, 86% or higher, 88% or higher, 90% or higher, 92% orhigher, 94% or higher, 96% or higher, 98% or higher, or 99% or higher,sequence identity to the amino acid sequence represented by SEQ ID NO:70 (FIG. 78) of the Sequence Listing and is carried by a polypeptidehaving FGF binding activity;(c) an amino acid sequence that is derived from the amino acid sequencerepresented by SEQ ID NO: 70 (FIG. 78) of the Sequence Listing by thesubstitution, deletion, addition, or insertion of 1 to 50, 1 to 45, 1 to40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid(s) and is carried bya polypeptide having FGF binding activity; and(d) an amino acid sequence that is encoded by the nucleotide sequence ofa nucleotide hybridizing under stringent conditions to a nucleotidehaving a nucleotide sequence complementary to a nucleotide sequenceencoding the amino acid sequence represented by SEQ ID NO: 70 (FIG. 78)of the Sequence Listing and is carried by a polypeptide having FGFbinding activity.

The polypeptide described in any one of (b) to (d) may have FGFR2activities other than the FGF binding activity.

The FGFR2 IIIc protein according to the present invention comprises anamino acid sequence described in any one of the following (a) to (d)(hereinafter, referred to as an “FGFR2 IIIc amino acid sequence”),consists of an amino acid sequence comprising the FGFR2 IIIc amino acidsequence, or consists of the FGFR2 IIIc amino acid sequence:

(a) an amino acid sequence represented by SEQ ID NO: 71 (FIG. 79) of theSequence Listing;(b) an amino acid sequence that exhibits 80% or higher, 82% or higher,84% or higher, 86% or higher, 88% or higher, 90% or higher, 92% orhigher, 94% or higher, 96% or higher, 98% or higher, or 99% or higher,sequence identity to the amino acid sequence represented by SEQ ID NO:71 (FIG. 79) of the Sequence Listing and is carried by a polypeptidehaving FGF binding activity;(c) an amino acid sequence that is derived from the amino acid sequencerepresented by SEQ ID NO: 71 (FIG. 79) of the Sequence Listing by thesubstitution, deletion, addition, or insertion of 1 to 50, 1 to 45, 1 to40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid(s) and is carried bya polypeptide having FGF binding activity; and(d) an amino acid sequence that is encoded by the nucleotide sequence ofa nucleotide hybridizing under stringent conditions to a nucleotidehaving a nucleotide sequence complementary to a nucleotide sequenceencoding the amino acid sequence represented by SEQ ID NO: 71 (FIG. 79)of the Sequence Listing and is carried by a polypeptide having FGFbinding activity.

The polypeptide described in any one of (b) to (d) may have FGFR2activities other than the FGF binding activity.

Examples of the amino acid sequences of human FGFR2 IIIb and human FGFR2IIIc can include the amino acids (a) to (d) as well as the amino acidspublished under NP_(—)075259 and NP_(—)000132, respectively.

(iv) The FGFR2 protein of the present invention can be obtained fromFGFR2-expressing cells, tissues, or cancer tissues, cells derived fromthe tissues, cultures of the cells, and the like, of a vertebrate,preferably of a mammal, more preferably of a rodent such as a mouse or arat and a human, even more preferably of a human, a rat, or a mouse.

Examples of the normal tissues highly expressing FGFR2 can include thebrain, the large intestine, thyroid glands, the uterine, thegallbladder, and the skin. Gene amplification is found in some cancershighly expressing FGFR2, such as stomach cancer and breast cancer, whileoverexpression is found in some cancers highly expressing FGFR2, such aspancreatic cancer and ovarian cancer. Examples of the cultured celllines highly expressing FGFR2 IIIb can include stomach cancer cell linesand breast cancer cell lines. Examples of the cultured cell lines highlyexpressing FGFR2 IIIb can include colorectal (cecal) cancer cell lines.Examples of cancer tissues expressing FGFR2 IIIc can include tissueswith uterine cervix cancer and non-small cell lung cancer. Of thesecancers, uterine cervix cancer highly expresses FGFR2 IIIc.

The FGFR2 protein of the present invention may be a native(non-recombinant) or recombinant protein. The FGFR2 protein is alsomeant to include fusion products with another peptide or protein such asa carrier or a tag. The FGFR2 protein is further meant to include formsprovided with chemical modification including the addition of a polymersuch as PEG and/or with biological modification including sugar chainmodification. Moreover, the FGFR2 protein of the present invention ismeant to include an FGFR2 protein fragment. An FGFR2 protein fragmentpossessing the properties described above in (i) and/or (ii) is referredto as a functional fragment of the FGFR2 protein.

(2-2) Antigen Gene

The FGFR2 IIIb gene according to the present invention comprises anucleotide sequence described in any one of the following (a) to (c)(hereinafter, referred to as an “FGFR2 gene sequence”), consists of anucleotide sequence comprising the FGFR2 gene sequence, or consists ofthe FGFR2 gene sequence:

(a) a nucleotide sequence encoding the amino acid sequence representedby SEQ ID NO: 70 (FIG. 78) of the Sequence Listing;(b) a nucleotide sequence that hybridizes under stringent conditions toa nucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence encoding the amino acid sequence represented by SEQID NO: 70 (FIG. 78) of the Sequence Listing and encodes the amino acidsequence of a polypeptide having FGF binding activity; and(c) a nucleotide sequence that encodes an amino acid sequence derivedfrom the amino acid sequence represented by SEQ ID NO: 70 (FIG. 78) ofthe Sequence Listing by the substitution, deletion, addition, orinsertion of 1 to 50, 1 to 45, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 aminoacid(s) and encodes the amino acid sequence of a polypeptide having FGFbinding activity.

The polypeptide having the amino acid sequence encoded by the nucleotidesequence (b) or (c) may have FGFR2 activities other than the FGF bindingactivity.

The FGFR2 IIIc gene according to the present invention comprises anucleotide sequence described in any one of the following (a) to (c)(hereinafter, referred to as an “FGFR2 gene sequence”), consists of anucleotide sequence comprising the FGFR2 gene sequence, or consists ofthe FGFR2 gene sequence:

(a) a nucleotide sequence encoding the amino acid sequence representedby SEQ ID NO: 71 (FIG. 79) of the Sequence Listing;(b) a nucleotide sequence that hybridizes under stringent conditions toa nucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence encoding the amino acid sequence represented by SEQID NO: 71 (FIG. 79) of the Sequence Listing and encodes the amino acidsequence of a polypeptide having FGF binding activity; and(c) a nucleotide sequence that encodes an amino acid sequence derivedfrom the amino acid sequence represented by SEQ ID NO: 71 (FIG. 79) ofthe Sequence Listing by the substitution, deletion, addition, orinsertion of 1 to 50, 1 to 45, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 aminoacid(s) and encodes the amino acid sequence of a polypeptide having FGFbinding activity.

The polypeptide having the amino acid sequence encoded by the nucleotidesequence (b) or (c) may have FGFR2 activities other than the FGF bindingactivity.

The expression and expression level of the FGFR2 gene may be assayedwith either an FGFR2 gene transcript or the FGFR2 protein as an index.The former index can be determined by RT-PCR, Northern blothybridization, or the like, while the latter index can be determined by,for example, immunoassay such as enzyme-linked immunosorbent assay(hereinafter, referred to as “ELISA”), Western blotting, orimmunohistological staining.

(2-3) Preparation of Antigenic Protein

The FGFR2 protein of the present invention can be prepared bypurification or isolation from animal tissues (including body fluids),cells derived from the tissues, or cultures of the cells, generecombination, in vitro translation, chemical synthesis, etc.

(2-3-1) Purification or Isolation of Non-Recombinant FGFR2

The non-recombinant FGFR2 protein can be purified or isolated fromFGFR2-expressing cells, normal tissues, or cancer tissues, or cellsderived therefrom. Examples of the FGFR2-expressing normal tissues,cancer tissues, or cancer cells can include those described in (iv) ofparagraph (2-2), though the origin of the non-recombinant FGFR2 proteinis not limited thereto.

The purification or isolation from such tissues, cells, cell cultures,or the like, can be performed by the combination of approaches wellknown by those skilled in the art, such as fractionation andchromatography. Such approaches include, but are not limited to, saltingout, gel filtration, ion-exchange chromatography, affinitychromatography, hydrophobic chromatography, normal-phase orreverse-phase chromatography, and the like. A column for affinitychromatography can be prepared by packing the column with an affinitygel cross-linked with an anti-FGFR2 monoclonal antibody. A crude orpartially purified fraction containing the FGFR2 protein is applied tothis column. Subsequently, non-specifically adsorbed substances areremoved with sterilized phosphate-buffered saline (PBS), and a buffersolution for elution can then be applied thereto to thereby selectivelyrecover the FGFR2 protein. The solution containing the FGFR2 protein canbe subjected to gel filtration or to buffer replacement and/orconcentration using a concentrator such as Centriprep.

(2-3-2) Preparation of Recombinant FGFR2 Protein

The FGFR2 protein of the present invention can also be prepared in arecombinant form. Specifically, host cells are transfected with a geneencoding the amino acid sequence of the FGFR2 protein or an FGFR2protein fragment, and the FGFR2 protein can be recovered from culturesof the cells. For example, the FGFR2 gene or its fragment is insertedinto an expression vector. Subsequently, prokaryotic or eukaryotic hostcells are transfected with the resulting recombinant vector, and theobtained recombinant cells can be incubated to thereby express the FGFR2protein. An expression pattern known in the art, such as secretionexpression, intracellular expression of soluble forms, or expression ininclusion body forms can be used. Also, the FGFR2 protein can beexpressed not only as a molecule having the same amino terminus (Nterminus) and/or carboxy terminus (C terminus) as native ones, but alsoas a fusion protein with a secretory signal, an intracellularlocalization signal, a tag for affinity purification, or a partnerpeptide. The FGFR2 protein can be purified or isolated from suchrecombinant cell cultures by an appropriate combination of methods suchas fractionation and chromatography described in (2-3-1)

Purification or Isolation of Non-Recombinant FGFR2 Protein.

The FGFR2 gene or its fragment can be prepared by a method well known bythose skilled in the art.

Examples thereof can include: polymerase chain reaction (hereinafter,referred to as “PCR”; Saiki, R. K., et al., Science (1988) 239, p.487-489) with a cDNA library prepared from FGFR2-expressing cells,tissues, or the like as a template using one set of primers capable ofspecifically amplifying the sequence; reverse transcription PCR(hereinafter, referred to as “RT-PCR”) with an mRNA fraction preparedfrom FGFR2-expressing cells, tissues, or the like as a template using aprimer capable of reverse-transcribing the sequence and one set ofprimers capable of specifically amplifying the sequence; expressioncloning using immunoassay; and cDNA cloning using the partial amino acidsequence of purified FGFR2 protein.

(2-3-3) In Vitro Translation

The FGFR2 protein of the present invention can also be prepared by invitro translation. Such a translation method is not particularly limitedas long as the method employs a cell-free translation system involvingenzymes necessary for transcription and translation, substrates, andenergy substances. Examples thereof can include a method using RapidTranslation System (RTS) manufactured by Roche Diagnostics K.K.

(2-3-4) Chemical Synthesis

The FGFR2 protein of the present invention can also be prepared bychemical synthesis. Examples of the chemical synthesis method caninclude solid-phase peptide synthesis methods such as Fmoc and Bocsynthesis methods.

3. Antibody

(3-1) Classification of Antibody

The antibodies of the present invention may be either monoclonal orpolyclonal antibodies. Examples of the monoclonal antibody of thepresent invention can include non-human animal-derived antibodies(non-human animal antibodies), human-derived antibodies (humanantibodies), chimeric antibodies, and humanized antibodies.

Examples of the non-human animal antibody can include antibodies derivedfrom vertebrates such as mammals and birds. Examples of themammal-derived antibody can include rodent-derived antibodies such asmouse antibodies and rat antibodies. Examples of the bird-derivedantibody can include chicken antibodies. Examples of the anti-humanFGFR2 rat monoclonal antibody can include FR2-10, FR2-13, and FR2-14.

Examples of the chimeric antibody can include, but are not limited to,an antibody comprising non-human animal antibody-derived variableregions bound with human antibody (human immunoglobulin) constantregions. Examples of the chimeric antibody comprising non-human animalantibody-derived variable regions bound with human antibody constantregions can include cFR2-10, cFR2-13, and cFR2-14 having heavy and lightchain variable regions derived from the rat monoclonal antibody FR2-10,FR2-13, or FR2-14 mentioned above, and human heavy and light chainconstant regions.

Examples of the humanized antibody can include, but are not limited to,a human antibody (human immunoglobulin variable regions) grafted withCDRs in the variable regions of a non-human animal antibody, a humanantibody grafted with the CDRs as well as with partial sequences offramework regions of a non-human animal antibody, and an antibody havinghuman antibody amino acid(s) substituted for one or two or morenon-human animal antibody-derived amino acid(s) in any of thesehumanized antibodies. Examples of the CDRs in the variable regions of anon-human animal antibody can include CDRH1 to CDRH3 in the heavy chainvariable region and CDRL1 to CDRL3 in the light chain variable regionderived from FR2-10, FR2-13, or FR2-14 mentioned above.

The human antibody is not particularly limited as long as the antibodyrecognizes the antigen of the present invention. Examples thereof caninclude a human antibody binding to the same site, as in the case of anantibody having the CDRs of the antibody of the present invention, and ahuman antibody binding to the same site on FGFR2 as in the case ofFR2-10, FR2-13, or FR2-14 mentioned above.

The antibody according to the present invention may be comprised ofportions derived from a plurality of different antibodies as long as theantibody has FGFR2 binding activity. Examples of such an antibody caninclude an antibody comprising heavy and/or light chains exchanged amonga plurality of different antibodies, an antibody comprising full-lengthheavy and/or light chains exchanged thereamong, an antibody comprisingvariable or constant regions exchanged thereamong, and an antibodycomprising all or some CDRs exchanged thereamong. The heavy and lightchain variable regions of the chimeric antibody may be derived fromdifferent antibodies of the present invention. CDRH1 to CDRH3 and CDRL1to CDRL3 in the heavy and light chain variable regions of the humanizedantibody may be derived from two or more different antibodies of thepresent invention. CDRH1 to CDRH3 and CDRL1 to CDRL3 in the heavy andlight chain variable regions of the human antibody may be a combinationof CDRs carried by two or more different antibodies of the presentinvention. Such an antibody comprised of portions derived from aplurality of different antibodies may have one or two or more of theactivities described in paragraphs (3-3) to (3-6).

Examples of the isotype of the monoclonal antibody of the presentinvention can include, but are not particularly limited to, IgG such asIgG1, IgG2, IgG3, and IgG4, IgM, IgA such as IgA1 and IgA2, IgD, and IgEand can preferably include IgG and IgM. The isotype and subclass of themonoclonal antibody can be determined by, for example, an Ouchterlonytest, ELISA, or radio immunoassay (hereinafter, referred to as “RIA”). Acommercially available kit for identification (e.g., Mouse Typer Kit;Bio-Rad Laboratories, Inc., and RAT MONOCLONAL ANTIBODY ISOTYPING TESTKIT: AbD Serotec) may be used.

(3-2) Binding Specificity of Antibody

The antibody of the present invention recognizes the FGFR2 protein. Inother words, the antibody of the present invention binds to the FGFR2protein. Such an antibody is referred to as an “anti-FGFR2 antibody”.Preferably, the antibody of the present invention specificallyrecognizes the FGFR2 protein. In other words, preferably, the antibodyof the present invention specifically binds to the FGFR2 protein. Morepreferably, the antibody of the present invention specifically binds tothe FGFR2 IIIb protein and/or the FGFR2 IIIc protein. Even morepreferably, the antibody of the present invention specifically binds tothe immunoglobulin-like domain (hereinafter, referred to as “Ig-likedomain”) of the FGFR2 IIIb protein and/or the FGFR2 IIIc protein.Examples of such an Ig-like domain can include Ig-like domain 2 andIg-like domain 3.

In the present invention, the “specific recognition”, i.e., “specificbinding”, means binding which is not non-specific adsorption. Examplesof criteria for determination of whether binding is specific or not caninclude a dissociation constant (hereinafter, referred to as “KD”).Preferably, the antibody of the present invention has a KD value of1×10⁻⁵ or lower, 5×10⁻⁶ or lower, 2×10⁻⁶ or lower, or 1×10⁻⁶ or lower,more preferably 5×10⁻⁷ or lower, 2×10⁻⁷ or lower, or 1×10⁻⁷ or lower,even more preferably 5×10⁻⁸ or lower, 2×10⁻⁸ or lower, or 1×10⁻⁸ orlower, further more preferably 5×10⁻⁹ or lower, 2×10⁻⁹ or lower, or1×10⁻⁹ or lower, most preferably 5×10⁻¹⁰ or lower, 2×10⁻¹⁰ or lower, or1×10⁻¹⁰ or lower for the FGFR2 protein.

In the present invention, the binding of the antibody to the antigen canbe assayed or determined by ELISA, RIA, surface plasmon resonance(hereinafter, referred to as “SPR”) analysis, or the like. Examples ofequipment used in the SPR analysis can include BIAcore™ (manufactured byGE Healthcare Bio-Sciences Corp.), ProteOn™ (manufactured by Bio-RadLaboratories, Inc.), SPR-Navi™ (manufactured by BioNavis Oy Ltd.),Spreeta™ (manufactured by Texas Instruments Inc.), SPRi-Plex II™(manufactured by Horiba, Ltd.), and Autolab SPR™ (manufactured byMetrohm Japan Ltd.). The binding of the antibody to the antigenexpressed on cell surface can be assayed by flow cytometry, Cell-ELISA,or the like.

(3-3) Antitumor Activity of Antibody

The antibody of the present invention has antitumor activity.Preferably, the antibody of the present invention has antitumor activityin vivo. In the present invention, the “antitumor activity” has the samemeaning as “anti-cancer activity”.

In the present invention, the antitumor activity means the activity ofinhibiting the growth, malignant transformation, invasion, or metastasisof tumor tissues and/or tumor cells, increase in tumor size or weight,etc.

The antitumor activity can be evaluated according to a standard method.The in vivo antitumor activity can be evaluated as an effect on humantumor, for example, by use of human cancer tissue- or cancercell-transplanted non-human animal models (xenografts). Examples of thenon-human animal used for the xenografts can include mice such as nudemice, and rats.

Alternatively, the antitumor activity may be evaluated as suppressive orinhibitory activity against the growth of cancer cells.

(3-4) Cytotoxic Activity of Antibody

The anti-FGFR2 antibody of the present invention may have antibodydependent cellular cytotoxic (ADCC) activity and/or complement dependentcytotoxic (CDC) activity and/or antibody dependent cell phagocytosis(ADCP) activity. Preferably, the antibody of the present invention hasADCC activity. More preferably, the antibody of the present inventionhas ADCC activity against FGFR2-expressing cells. The ADCC activity, theCDC activity, and the ADCP activity can be assayed by a method known inthe art.

Cells expressing the antigen of interest (target cells) and effectorcells capable of killing the target cells are used in the ADCC activityassay. The effector cells recognize the Fc regions of antibodies boundwith the target cells via Fcγ receptors. The effector cells kill thetarget cells by signals transduced from the Fcγ receptors. In the caseof assaying the ADCC activity of an antibody having a human-derived Fcregion, human NK cells are used as the effector cells. The human NKcells can be prepared from human peripheral blood mononuclear cells(PBMCs) by a method known in the art. Alternatively, PBMCs may be useddirectly as the effector cells.

Cells expressing the antigen of interest (target cells) and effectorcells (e.g., monocytes or macrophages) capable of englobing the targetcells are used in the ADCP activity assay. These effector cells can beprepared by inducing, by a method known in the art, differentiation frommonocyte fractions to macrophages, wherein said monocyte fractions havebeen separated from human peripheral blood mononuclear cells (PBMCs) bya method known in the art.

(3-5) Effect of Antibody on Signal Transduction

The biological activities and properties of the anti-FGFR2 antibody ofthe present invention can also be evaluated through FGFR2-mediated FGFsignals. Examples of the FGFR2-mediated signal transduction by FGFstimulation can include, but are not particularly limited to, FGFR2autophosphorylation, recruitment of FGFR substrates and promotionthereof, and activation of signaling pathways such as MAPK, PI3K, Akt,and extracellular signal-regulated kinase (ERK) pathways via theseevents. Examples of the FGFR substrates can include FGFR substrate 2a(FRS2a). Testing methods for evaluating the activation of this signaltransduction and the inhibition thereof are not particularly limited andcan be arbitrarily selected from methods known in the art. Examplesthereof can include evaluation systems for ERK signal transduction, andElk1 luciferase reporter assay described later.

Preferably, the antibody of the present invention also has neutralizingactivity against FGFR2. More preferably, the antibody of the presentinvention has neutralizing activity against FGFR2 IIIb and/or FGFR2IIIc. The neutralizing activity means the activity of inhibiting orsuppressing the activation of FGFR2 by an FGFR2 ligand. For example, anantibody inhibiting FGF dependent FGFR2-mediated signals, signaltransduction, or the like can be confirmed to have such neutralizingactivity. Exemplary assay of the neutralizing activity is shown in 2)-3of Example 2 and Example 11.

(3-6) Activity of Inhibiting Receptor-Ligand Binding by Antibody

Preferably, the antibody of the present invention inhibits the bindingof FGFR2 to its ligand. More preferably, the antibody of the presentinvention inhibits the binding of FGFR2 IIIb and/or FGFR2 IIIc to FGF.This inhibition of receptor-ligand binding may be any of competitiveinhibition and noncompetitive inhibition. Examples of the ligands ofFGFR2 IIIb and FGFR2 IIIc can include FGF1, FGF3, FGF7, FGF10, FGF22,and FGF23, and FGF1, FGF2, FGF4, FGF6, FGF9, FGF17, FGF18, FGF21, andFGF23, respectively.

(3-7) Monoclonal Antibody

The present invention provides a monoclonal antibody. The monoclonalantibody includes, for example, non-human animal-derived monoclonalantibodies such as rat, mouse, rabbit, chicken, and fish antibodies,chimeric antibodies, humanized antibodies, human antibodies, functionalfragments thereof, and modified forms of these antibodies or functionalfragments. Of them, examples of the rat monoclonal antibody can includethe FR2-10, FR2-13, and FR2-14 antibodies.

FR2-10 is an anti-human FGFR2 rat monoclonal antibody obtained by themethod described in Example 1. The nucleotide sequence of the heavychain variable region of FR2-10 is described in SEQ ID NO: 11 (FIG. 19)of the Sequence Listing, and its amino acid sequence is described in SEQID NO: 12 (FIG. 20). The nucleotide sequence of the light chain variableregion of FR2-10 is described in SEQ ID NO: 20 (FIG. 28) of the SequenceListing, and its amino acid sequence is described in SEQ ID NO: 21 (FIG.29). The amino acid sequence of CDRH1 of FR2-10 is described in SEQ IDNO: 52 (FIG. 60). The amino acid sequence of CDRH2 thereof is describedin SEQ ID NO: 53 (FIG. 61). The amino acid sequence of CDRH3 thereof isdescribed in SEQ ID NO: 54 (FIG. 62). The amino acid sequence of CDRL1thereof is described in SEQ ID NO: 61 (FIG. 69). The amino acid sequenceof CDRL2 thereof is described in SEQ ID NO: 62 (FIG. 70). The amino acidsequence of CDRL3 thereof is described in SEQ ID NO: 63 (FIG. 71).

FR2-13 is an anti-human FGFR2 rat monoclonal antibody obtained by themethod described in Example 1. The nucleotide sequence of the heavychain variable region of FR2-13 is described in SEQ ID NO: 13 (FIG. 21)of the Sequence Listing, and its amino acid sequence is described in SEQID NO: 14 (FIG. 22). The nucleotide sequence of the light chain variableregion of FR2-13 is described in SEQ ID NO: 23 (FIG. 31) of the SequenceListing, and its amino acid sequence is described in SEQ ID NO: 24 (FIG.32). The amino acid sequence of CDRH1 of FR2-13 is described in SEQ IDNO: 55 (FIG. 63). The amino acid sequence of CDRH2 thereof is describedin SEQ ID NO: 56 (FIG. 64). The amino acid sequence of CDRH3 thereof isdescribed in SEQ ID NO: 57 (FIG. 65). The amino acid sequence of CDRL1thereof is described in SEQ ID NO: 64 (FIG. 72). The amino acid sequenceof CDRL2 thereof is described in SEQ ID NO: 65 (FIG. 73). The amino acidsequence of CDRL3 thereof is described in SEQ ID NO: 66 (FIG. 74).

FR2-14 is an anti-human FGFR2 rat monoclonal antibody obtained by themethod described in Example 1. The nucleotide sequence of the heavychain variable region of FR2-14 is described in SEQ ID NO: 15 (FIG. 23)of the Sequence Listing, and its amino acid sequence is described in SEQID NO: 16 (FIG. 24). The nucleotide sequence of the light chain variableregion of FR2-14 is described in SEQ ID NO: 25 (FIG. 33) of the SequenceListing, and its amino acid sequence is described in SEQ ID NO: 26 (FIG.34). The amino acid sequence of CDRH1 of FR2-14 is described in SEQ IDNO: 58 (FIG. 66). The amino acid sequence of CDRH2 thereof is describedin SEQ ID NO: 59 (FIG. 67). The amino acid sequence of CDRH3 thereof isdescribed in SEQ ID NO: 60 (FIG. 68). The amino acid sequence of CDRL1thereof is described in SEQ ID NO: 67 (FIG. 75). The amino acid sequenceof CDRL2 thereof is described in SEQ ID NO: 68 (FIG. 76). The amino acidsequence of CDRL3 thereof is described in SEQ ID NO: 69 (FIG. 77).

The antibody mutant of the present invention preferably exhibits, forexample, reduced sensitivity to protein degradation or oxidation, animproved biological activity, an improved ability to bind to theantigen, or physicochemical or functional properties imparted thereto.Examples of such an antibody mutant can include an antibody having anamino acid sequence derived from the amino acid sequence of the originalantibody by conservative amino acid substitution. The conservative aminoacid substitution is a substitution that occurs in an amino acid grouprelated to amino acid side chains.

Preferred amino acid groups are as follows: an acidic group includingaspartic acid and glutamic acid; a basic group including lysine,arginine, and histidine; a nonpolar group including alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan;and an uncharged polar family including glycine, asparagine, glutamine,cysteine, serine, threonine, and tyrosine. Other preferred amino acidgroups are as follows: an aliphatic hydroxy group including serine andthreonine; an amide-containing group including asparagine and glutamine;an aliphatic group including alanine, valine, leucine, and isoleucine;and an aromatic group including phenylalanine, tryptophan, and tyrosine.Such amino acid substitution in the antibody mutant is preferablyperformed without reducing the antigen binding activity of the originalantibody.

Aspartic acid contained in a protein is easily converted to isoasparticacid by isomerization when an amino acid linked thereto on the Cterminal side has a small side chain. On the other hand, asparagine iseasily converted to aspartic acid by deamidation and may be furtherconverted to isoaspartic acid by isomerization. The progression of suchisomerization or deamidation may influence the stability of the protein.Accordingly, aspartic acid or asparagine in the protein or, for example,an amino acid adjacent thereto, can be substituted by a different aminoacid in order to circumvent such isomerization or deamidation.Preferably, an antibody mutant having such amino acid substitutionmaintains the antigen binding activity of the original antibody.

The present invention also encompasses, for example: an antibody mutanthaving an amino acid sequence derived from the amino acid sequence ofFR2-10, FR2-13, or FR2-14 of the present invention by conservative aminoacid substitution; and a mouse antibody, a rat antibody, a chimericantibody, a humanized antibody, or a human antibody comprising a CDRhaving an amino acid sequence in which a conservative amino acidmutation occurs in the amino acid sequence of any of CDRH1 to CDRH3 andCDRL1 to CDRL3 derived from FR2-10, FR2-13, or FR2-14.

The mutant of the antibody of the present invention encompasses a humanFGFR2-binding antibody mutant comprising CDRH1 to CDRH3 and CDRL1 toCDRL3 having amino acid sequences derived from the amino acid sequencesof any one or two or more of CDRH1 to CDRH3 and CDRL1 to CDRL3 derivedfrom FR2-10, FR2-13, or FR2-14 of the present invention by thesubstitution of 1 to several, preferably 1 to 3, more preferably 1 or 2,most preferably 1 amino acid(s) by different amino acid(s).

Preferred examples of the mutant of FR2-14 can include an antibodycomprising CDRH3 having an amino acid sequence derived from the CDRH3amino acid sequence by the substitution of amino acid(s) by differentamino acid(s).

Preferred examples of the mutant of FR2-14 in which amino acid(s) ofheavy chain CDRH3 is substituted can include: a mutant with the firstamino acid aspartic acid (amino acid at position 118 of SEQ ID NO: 51(FIG. 59)) substituted by glutamic acid; a mutant with the second aminoacid glycine (amino acid at position 119 of SEQ ID NO: 51 (FIG. 59))substituted by tyrosine, alanine, tryptophan, valine, arginine,asparagine, methionine, leucine, lysine, isoleucine, histidine,phenylalanine, or glutamic acid; and a mutant with the 8th amino acidthreonine substituted by alanine.

The antibody mutant also includes an antibody having CDRH1 to CDRH3 andCDRL1 to CDRL3 derived from a plurality of antibodies. Examples of sucha mutant can include an antibody mutant comprising CDRH3 derived from acertain antibody and CDRH1, CDRH2, and CDRL1 to CDRL3 derived fromanother antibody.

The “antibody” according to the present invention also encompasses theseantibody mutants.

The constant regions of the antibody of the present invention are notparticularly limited. Preferably, constant regions derived from a humanantibody are used in the antibody of the present invention for thetreatment or prevention of a disease in a human. Examples of the heavychain constant region of the human antibody can include Cγ1, Cγ2, Cγ3,Cγ4, Cμ, Cδ, Cα1, Cα2, and CE. Examples of the light chain constantregion of the human antibody can include Cκ and Cλ.

(3-8) Chimeric Antibody

The anti-FGFR2 chimeric antibody of the present invention or afunctional fragment thereof has antitumor activity. Preferably, theanti-FGFR2 chimeric antibody of the present invention or the functionalfragment thereof has antitumor activity in vivo. Also preferably, such achimeric antibody or a functional fragment thereof specifically binds tothe FGFR2 IIIb protein and/or the FGFR2 IIIc protein. More preferably,the chimeric antibody or functional fragment thereof binds to theIg-like domains of these proteins. Preferably, such a chimeric antibodyfurther has ADCC activity and/or ADCP activity. The chimeric antibody ofthe present invention or the functional fragment thereof also hasneutralizing activity against FGFR2. Preferably, the chimeric antibodyof the present invention or the functional fragment thereof hasneutralizing activity against FGFR2 IIIb and/or FGFR2 IIIc. Morepreferably, the chimeric antibody of the present invention or thefunctional fragment thereof has neutralizing activity against FGFR2 IIIband FGFR2 IIIc. Preferably, the chimeric antibody of the presentinvention or the functional fragment thereof further inhibits thebinding of FGFR2 to its ligand.

The nucleotide sequence and amino acid sequence of the light chain ofcFR2-10 exemplified as the rat-human chimeric antibody of the presentinvention and the nucleotide sequence and amino acid sequence of theheavy chain thereof are shown in SEQ ID NOs: 31, 32, 35, and 36 (FIGS.39, 40, 43, and 44), respectively, of the Sequence Listing. Likewise,the nucleotide sequence and amino acid sequence of the light chain ofcFR2-13 and the nucleotide sequence and amino acid sequence of the heavychain thereof are shown in SEQ ID NOs: 39, 40, 43, and 44 (FIGS. 47, 48,51, and 52), respectively, of the Sequence Listing. The nucleotidesequence and amino acid sequence of the light chain of cFR2-14 and thenucleotide sequence and amino acid sequence of the heavy chain thereofare shown in SEQ ID NOs: 47, 48, 50, and 51 (FIGS. 55, 56, 58, and 59),respectively, of the Sequence Listing. Nucleotide positions 1 to 60 inthe nucleotide sequences of the light chains and amino acid positions 1to 20 in the amino acid sequences of the light chains represent a signalsequence, which is usually not contained in the nucleotide sequences andamino acid sequences of most of mature light chains, respectively.Likewise, nucleotide positions 1 to 57 in the nucleotide sequences ofthe heavy chains and amino acid positions 1 to 19 in the amino acidsequences of the heavy chains represent a signal sequence, which isusually not contained in the nucleotide sequences and amino acidsequences of most of mature heavy chains, respectively.

(3-9) Functional Fragment of Antibody

According to one aspect, the present invention provides a functionalfragment of the anti-FGFR2 antibody of the present invention. Thefunctional fragment of the antibody means a fragment that maintains atleast a portion of the functions of the antibody. Examples of suchfunctions of the antibody can generally include antigen bindingactivity, antigen activity-regulating activity, antibody dependentcellular cytotoxic (ADCC) activity, and antibody dependent cellphagocytosis (ADCP) activity. Examples of the functions of theanti-FGFR2 antibody of the present invention can include FGFR2 proteinbinding activity, ADCC activity, ADCP activity, neutralizing activityagainst FGFR2, in vivo antitumor activity, and the activity ofinhibiting the binding of FGFR2 to its ligand.

The functional fragment of the antibody is not particularly limited aslong as the fragment of the antibody maintains at least a portion of theactivities of the antibody. Examples thereof can include, but are notlimited to, Fab, F(ab′)2, Fv, single chain Fv (scFv) comprising heavyand light chain Fvs linked via an appropriate linker, diabodies, linearantibodies, multispecific antibodies formed from antibody fragments, andFab′, which is a monovalent fragment of antibody variable regionsobtained by the treatment of F(ab′)2 under reducing conditions. Thefunctional fragment of the antibody of the present invention is alsomeant to include a molecule comprising the fragment of the antibody ofthe present invention as well as other portions, such as scFv retaininga linker portion.

A molecule that is derived from the antibody protein by the deletion of1 to several or more amino acid(s) at its amino terminus and/or carboxyterminus and maintains at least a portion of the functions of theantibody is also encompassed in the meaning of the functional fragmentof the antibody. For example, the heavy chain of an antibody produced bycultured mammalian cells is known to lack a lysine residue at thecarboxy terminus (Journal of Chromatography A, 705: 129-134 (1995)).Also, the heavy chain of such an antibody is known to lack two aminoacid residues (glycine and lysine) at the carboxy terminus and insteadhave an amidated proline residue at the carboxy terminus (AnalyticalBiochemistry, 360: 75-83 (2007)). The deletion and the modification inthese heavy chain sequences, however, do not influence the ability ofthe antibody to bind to the antigen or its effector functions(complement activation, antibody dependent cytotoxic effects, etc.).Such a modified form of the functional fragment of the antibody is alsoencompassed by the antibody of the present invention or the functionalfragment thereof, or a modified form (described later) of the antibodyor functional fragment.

The antibody of the present invention or the functional fragment thereofmay be a multispecific antibody having specificity for at least 2 typesof different antigens. The multispecific antibody is not limited to abispecific antibody, which binds to 2 types of different antigens, andan antibody having specificity for 3 or more types of different antigensis also encompassed in the meaning of the “multispecific antibody” ofthe present invention.

The multispecific antibody of the present invention may be a full-lengthantibody or a functional fragment thereof (e.g., bispecific F(ab′)2antibody). The bispecific antibody can also be prepared by linking theheavy and light chains (HL pairs) of two types of antibodies.Alternatively, the bispecific antibody may be obtained by fusing two ormore types of monoclonal antibody-producing hybridomas to preparebispecific antibody-producing fusion cells (Millstein et al., Nature(1983) 305, p. 537-539). The multispecific antibody can also be preparedin the same way as above.

According to one aspect, the antibody of the present invention is asingle chain antibody (single chain Fv; hereinafter, referred to as“scFv”). The scFv is obtained by linking the heavy and light chain Vregions of the antibody via a polypeptide linker (Pluckthun, ThePharmacology of Monoclonal Antibodies, 113, Rosenburg and Moore, ed.,Springer Verlag, New York, p. 269-315 (1994); and Nature Biotechnology(2005), 23, p. 1126-1136). Also, bi-scFv comprising two scFvs linked viaa polypeptide linker can be used as a bispecific antibody.Alternatively, multi-scFv comprising three or more scFvs may be used asa multispecific antibody.

The present invention includes a single chain immunoglobulin comprisingfull-length heavy and light chain sequences of the antibody linked viaan appropriate linker (Lee, H-S, et al., Molecular Immunology (1999),36, p. 61-71; and Shirrmann, T. et al., mAbs (2010), 2 (1) p. 1-4). Sucha single chain immunoglobulin can be dimerized to thereby maintain astructure and activities similar to those of the antibody, which isoriginally a tetramer. Also, the antibody of the present invention maybe an antibody that has a single heavy chain variable region and has nolight chain sequence. Such an antibody, called a single domain antibody(sdAb) or a nanobody, has been reported to maintain the ability to bindto an antigen (Muyldemans S. et al., Protein Eng. (1994), 7 (9),1129-35; and Hamers-Casterman C. et al., Nature (1993), 363 (6428),446-8). These antibodies are also encompassed in the meaning of thefunctional fragment of the antibody according to the present invention.

(3-10) Humanized Antibody and Human Antibody

According to one aspect, the present invention provides a humanizedantibody or a functional fragment thereof.

The anti-FGFR2 humanized antibody of the present invention or afunctional fragment thereof has antitumor activity. Preferably, theanti-FGFR2 humanized antibody of the present invention or the functionalfragment thereof has antitumor activity in vivo. Also preferably, such ahumanized antibody or a functional fragment thereof specifically bindsto the FGFR2 IIIb protein and/or the FGFR2 IIIc protein. Morepreferably, the humanized antibody or functional fragment thereof bindsto the Ig-like domains of these proteins. Preferably, such a humanizedantibody or a functional fragment thereof further has ADCC activityand/or ADCP activity. The humanized antibody of the present invention orthe functional fragment thereof also has neutralizing activity againstFGFR2. Preferably, the humanized antibody of the present invention orthe functional fragment thereof has neutralizing activity against FGFR2IIIb and/or FGFR2 IIIc. More preferably, the humanized antibody of thepresent invention or the functional fragment thereof has neutralizingactivity against FGFR2 IIIb and FGFR2 IIIc. Preferably, the humanizedantibody of the present invention or the functional fragment thereoffurther inhibits the binding of FGFR2 to its ligand.

Preferred examples of the humanized antibody of the present inventioncan include humanized antibodies having the heavy chain CDRH1 to CDRH3and light chain CDRL1 to CDRL3 of the rat FR2-10 antibody, the ratFR2-13 antibody, or the rat FR2-14 antibody as described below in A toC.

(A. Humanized Antibody Having Heavy Chain CDRH1 to CDRH3 and Light ChainCDRL1 to CDRL3 of Rat FR2-10 Antibody)

Examples of the anti-FGFR2 humanized antibody of the present inventionor a functional fragment thereof can include a humanized antibody thatconsists of a heavy chain having a variable region comprising CDRH1consisting of the amino acid sequence represented by SEQ ID NO: 52 (FIG.60) of the Sequence Listing, CDRH2 consisting of the amino acid sequencerepresented by SEQ ID NO: 53 (FIG. 61) of the Sequence Listing, andCDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 54(FIG. 62) of the Sequence Listing, and a light chain having a variableregion comprising CDRL1 consisting of the amino acid sequencerepresented by SEQ ID NO: 61 (FIG. 69) of the Sequence Listing, CDRL2consisting of the amino acid sequence represented by SEQ ID NO: 62 (FIG.70) of the Sequence Listing, and CDRL3 consisting of the amino acidsequence represented by SEQ ID NO: 63 (FIG. 71) of the Sequence Listing,and that recognizes the FGFR2 protein of the present invention, and afragment of the antibody that maintains the FGFR2 protein bindingactivity of the antibody, and mutants of the antibody or the fragment.

(B. Humanized Antibody Having Heavy Chain CDRH1 to CDRH3 and Light ChainCDRL1 to CDRL3 of Rat FR2-13 Antibody)

Alternative examples of the anti-FGFR2 humanized antibody or afunctional fragment thereof can include a humanized antibody thatconsists of a heavy chain having a variable region comprising CDRH1consisting of the amino acid sequence represented by SEQ ID NO: 55 (FIG.63) of the Sequence Listing, CDRH2 consisting of the amino acid sequencerepresented by SEQ ID NO: 56 (FIG. 64) of the Sequence Listing, andCDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 57(FIG. 65) of the Sequence Listing, and a light chain having a variableregion comprising CDRL1 consisting of the amino acid sequencerepresented by SEQ ID NO: 64 (FIG. 72) of the Sequence Listing, CDRL2consisting of the amino acid sequence represented by SEQ ID NO: 65 (FIG.73) of the Sequence Listing, and CDRL3 consisting of the amino acidsequence represented by SEQ ID NO: 66 (FIG. 74) of the Sequence Listing,and that recognizes the FGFR2 protein of the present invention, and afragment of the antibody that maintains the FGFR2 protein bindingactivity of the antibody, and mutants thereof.

(C. Humanized Antibody Having Heavy Chain CDRH1 to CDRH3 and Light ChainCDRL1 to CDRL3 of Rat FR2-14 Antibody)

Alternative examples of the anti-FGFR2 humanized antibody or afunctional fragment thereof can include a humanized antibody thatconsists of a heavy chain having a variable region comprising CDRH1consisting of the amino acid sequence represented by SEQ ID NO: 58 (FIG.66) of the Sequence Listing, CDRH2 consisting of the amino acid sequencerepresented by SEQ ID NO: 59 (FIG. 67) of the Sequence Listing, andCDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 60(FIG. 68) of the Sequence Listing, and a light chain having a variableregion comprising CDRL1 consisting of the amino acid sequencerepresented by SEQ ID NO: 67 (FIG. 75) of the Sequence Listing, CDRL2consisting of the amino acid sequence represented by SEQ ID NO: 68 (FIG.76) of the Sequence Listing, and CDRL3 consisting of the amino acidsequence represented by SEQ ID NO: 69 (FIG. 77) of the Sequence Listing,and that recognizes the FGFR2 protein of the present invention, and afragment of the antibody that maintains the FGFR2 protein bindingactivity of the antibody, and mutants thereof.

The preferred humanized antibody of the present invention is not limitedto those described above in A to C. The humanized antibody is morepreferably a humanized FR2-14 antibody and its mutants. Examples thereofinclude, but are not limited to, hFR2-14_H1/L1 to hFR2-14_H19/L1. Themore preferred humanized antibody of the present invention alsoincludes, for example, an antibody comprising a heavy chain comprisingthe heavy chain variable region of any one of the humanized antibodieshFR2-14_H1/L1 to hFR2-14_H19/L1, and a light chain comprising the lightchain variable region of any one of the humanized antibodieshFR2-14_H1/L1 to hFR2-14_H19/L1.

hFR2-14_H1/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 74 (FIG. 82),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 75 (FIG. 83). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2, FGFR2 ligand dependent FGFR2 signal-neutralizingactivity, ADCC activity, and in vivo antitumor activity (see Examples).

hFR2-14_H2/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 76 (FIG. 84),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 77 (FIG. 85). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2, FGFR2 ligand dependent FGFR2 signal-neutralizingactivity, ADCC activity, and in vivo antitumor activity (see Examples).

hFR2-14_H3/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 78 (FIG. 86),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 79 (FIG. 87). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2, FGFR2 ligand dependent FGFR2 signal-neutralizingactivity, ADCC activity, and in vivo antitumor activity (see Examples).

hFR2-14_H4/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 80 (FIG. 88),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 81 (FIG. 89). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2, FGFR2 ligand dependent FGFR2 signal-neutralizingactivity, ADCC activity, and in vivo antitumor activity (see Examples).

hFR2-14_H5/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 82 (FIG. 90),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 83 (FIG. 91). The antibody was excellent in conformationalstability because of its high Tm value, had high binding activityagainst FGFR2, excellent thermal stability, FGFR2 ligand dependent FGFR2signal-neutralizing activity, and in vivo antitumor activity, andmaintained its high antigen binding activity even when exposed to severeconditions (see Examples).

hFR2-14_H6/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 84 (FIG. 92),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 85 (FIG. 93). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2 and FGFR2 ligand dependent FGFR2 signal-neutralizingactivity (see Examples).

hFR2-14_H7/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 86 (FIG. 94),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 87 (FIG. 95). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2 and FGFR2 ligand dependent FGFR2 signal-neutralizingactivity (see Examples).

hFR2-14_H8/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 88 (FIG. 96),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 89 (FIG. 97). The antibody was excellent in conformationalstability because of its high Tm value, had high binding activityagainst FGFR2, excellent thermal stability, FGFR2 ligand dependent FGFR2signal-neutralizing activity, ADCC activity, and in vivo antitumoractivity, and maintained its high antigen binding activity even whenexposed to severe conditions (see Examples).

hFR2-14_H9/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 90 (FIG. 98),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 91 (FIG. 99). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2, excellent thermal stability, FGFR2 ligand dependent FGFR2signal-neutralizing activity, and in vivo antitumor activity (seeExamples).

hFR2-14_H10/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 92 (FIG. 100),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 93 (FIG. 101). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2 and FGFR2 ligand dependent FGFR2 signal-neutralizingactivity (see Examples).

hFR2-14_H11/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 94 (FIG. 102),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 95 (FIG. 103). The antibody was excellent in conformationalstability because of its high Tm value, had high binding activityagainst FGFR2, excellent thermal stability, FGFR2 ligand dependent FGFR2signal-neutralizing activity, ADCC activity, and in vivo antitumoractivity, and maintained its high antigen binding activity even whenexposed to severe conditions (see Examples).

hFR2-14_H12/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 96 (FIG. 104),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 97 (FIG. 105). The antibody was excellent in conformationalstability because of its high Tm value, had high binding activityagainst FGFR2, excellent thermal stability, FGFR2 ligand dependent FGFR2signal-neutralizing activity, ADCC activity, ADCP activity, the activityof inhibiting the binding of an FGFR2 ligand to FGFR2, and in vivoantitumor activity, and maintained its high antigen binding activityeven when exposed to severe conditions (see Examples).

hFR2-14_H13/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 98 (FIG. 106),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 99 (FIG. 107). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2 and FGFR2 ligand dependent FGFR2 signal-neutralizingactivity (see Examples).

hFR2-14_H14/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 100 (FIG. 108),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 101 (FIG. 109). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2 and FGFR2 ligand dependent FGFR2 signal-neutralizingactivity (see Examples).

hFR2-14_H15/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 102 (FIG. 110),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 103 (FIG. 111). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2 and FGFR2 ligand dependent FGFR2 signal-neutralizingactivity (see Examples).

hFR2-14_H16/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 104 (FIG. 112),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 105 (FIG. 113). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2 and FGFR2 ligand dependent FGFR2 signal-neutralizingactivity (see Examples).

hFR2-14_H17/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 106 (FIG. 114),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 107 (FIG. 115). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2 and FGFR2 ligand dependent FGFR2 signal-neutralizingactivity (see Examples).

hFR2-14_H18/L1 is a humanized antibody obtained in Example 8. Thenucleotide sequence of the light chain of this antibody comprisesnucleotide positions 61 to 705 of SEQ ID NO: 72 (FIG. 80), and its aminoacid sequence comprises amino acid positions 21 to 235 of SEQ ID NO: 73(FIG. 81). The nucleotide sequence of the heavy chain of this antibodycomprises nucleotide positions 58 to 1401 of SEQ ID NO: 108 (FIG. 116),and its amino acid sequence comprises amino acid positions 20 to 467 ofSEQ ID NO: 109 (FIG. 117). The antibody was excellent in conformationalstability because of its high Tm value and had high binding activityagainst FGFR2, FGFR2 ligand dependent FGFR2 signal-neutralizingactivity, ADCC activity, and in vivo antitumor activity (see Examples).

hFR2-14_H19/L1 is a humanized antibody with regulated sugar chainmodification obtained in Example 9. The nucleotide sequence of the lightchain of this antibody comprises nucleotide positions 61 to 705 of SEQID NO: 72 (FIG. 80), and its amino acid sequence comprises amino acidpositions 21 to 235 of SEQ ID NO: 73 (FIG. 81). The nucleotide sequenceof the heavy chain of this antibody comprises nucleotide positions 58 to1401 of SEQ ID NO: 96 (FIG. 104), and its amino acid sequence comprisesamino acid positions 20 to 467 of SEQ ID NO: 97 (FIG. 105). The antibodywas excellent in conformational stability because of its high Tm value,had high binding activity against FGFR2, excellent thermal stability,FGFR2 ligand dependent FGFR2 signal-neutralizing activity, ADCCactivity, ADCP activity, the activity of inhibiting the binding of anFGFR2 ligand to FGFR2, and in vivo antitumor activity, and maintainedits high antigen binding activity even when exposed to severe conditions(see Examples).

These humanized FR2-14 antibodies were not found to cause weight loss orother significant toxic events, when administered to mice. ThehFR2-14_H12/L1 antibody and the hFR2-14_H19/L1 antibody wereadministered at a single dose of approximately 150 mg/kg to eachcynomolgus monkey and observed 14 days later. As a result, significantclinical findings, hematological change, weight loss, or othersignificant toxic events were not observed. Thus, the humanized antibodyof the present invention possesses excellent safety as a pharmaceuticalcomposition for treatment or prevention of a disease.

Among the more preferred humanized antibodies hFR2-14_H1/L1 tohFR2-14_H19/L1 of the present invention, the antibody is even morepreferably hFR2-14_H5/L1, hFR2-14_H11/L1, hFR2-14_H8/L1, hFR2-14_H12/L1,or hFR2-14_H19/L1, further more preferably hFR2-14_H12/L1 orhFR2-14_H19/L1.

The present invention also encompasses an antibody that comprises aheavy or light chain comprising an amino acid sequence having 80% orhigher, 82% or higher, 84% or higher, 86% or higher, 88% or higher, 90%or higher, 92% or higher, 94% or higher, 96% or higher, 98% or higher,or 99% or higher identity to the amino acid sequence of the heavy orlight chain of any one of the rat FR2-10, FR-10FR, and FR2-14antibodies, the chimeric cFR2-10, cFR2-13, and cFR2-14 antibodies, andthe humanized hFR2-14_H1/L1 to hFR2-14_H19/L1 antibodies of the presentinvention and binds to FGFR2, or a functional fragment thereof. Suchsequence identity is preferably 94% or higher, more preferably 96% orhigher, even more preferably 98% or higher, most preferably 99% orhigher. Preferably, these antibodies have one or more of the activitiesdescribed in paragraphs (3-3) to (3-6).

The identity or homology between two types of amino acid sequences canbe determined using the default parameter of Blast algorithm version2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer,Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997),“Gapped BLAST and PSI-BLAST: a new generation of protein database searchprograms”, Nucleic Acids Res. 25: 3389-3402). The Blast algorithm isalso available, for example, by Internet access athttp://blast.ncbi.nlm.nih.gov/.

The present invention also encompasses an antibody that comprises aheavy or light chain comprising an amino acid sequence derived from theamino acid sequence of the heavy or light chain of any one of the ratFR2-10, FR-10FR, and FR2-14 antibodies, the chimeric cFR2-10, cFR2-13,and cFR2-14 antibodies, and the humanized hFR2-14_H1/L1 tohFR2-14_H19/L1 antibodies of the present invention by the substitution,deletion, addition, or insertion of 1 to 50, 1 to 45, 1 to 40, 1 to 35,1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1to 4, 1 to 3, 1 or 2, or 1 amino acid(s) and binds to the FGFR2 protein,or a functional fragment thereof. Such an amino acid mutation ispreferably substitution. The number of mutated amino acids is preferably1 to 5, more preferably 1 to 4, even more preferably 1 to 3, furthermore preferably 1 or 2, most preferably 1. Preferably, these antibodieshave one or more of the activities described in paragraphs (3-3) to(3-6).

The present invention also encompasses an antibody that comprises aheavy or light chain comprising an amino acid sequence encoded by thenucleotide sequence of a nucleotide hybridizing under stringentconditions to a nucleotide having a nucleotide sequence complementary toa nucleotide sequence encoding the amino acid sequence of the heavy orlight chain of any one of the rat FR2-10, FR-10FR, and FR2-14antibodies, the chimeric cFR2-10, cFR2-13, and cFR2-14 antibodies, andthe humanized hFR2-14_H1/L1 to hFR2-14_H19/L1 antibodies of the presentinvention and binds to the FGFR2 protein, or a functional fragmentthereof. Preferably, these antibodies have one or more of the activitiesdescribed in paragraphs (3-3) to (3-6).

According to an alternative aspect, the present invention provides ahuman antibody or a functional fragment thereof. The human antibody ofthe present invention is not particularly limited as long as theantibody is derived from a human and binds to FGFR2. The human antibodyof the present invention or a functional fragment thereof has antitumoractivity, preferably in vivo antitumor activity. Also preferably, such ahuman antibody or a functional fragment thereof specifically binds tothe FGFR2 IIIb protein and/or the FGFR2 IIIc protein. More preferably,the human antibody or functional fragment thereof binds to the Ig-likedomains of these proteins. Preferably, such a human antibody or afunctional fragment thereof further has ADCC activity and/or ADCPactivity. The human antibody of the present invention or the functionalfragment thereof also has neutralizing activity against FGFR2.Preferably, the human antibody of the present invention or thefunctional fragment thereof has neutralizing activity against FGFR2 IIIband/or FGFR2 IIIc. More preferably, the human antibody of the presentinvention or the functional fragment thereof has neutralizing activityagainst FGFR2 IIIb and FGFR2 IIIc. Preferably, the human antibody of thepresent invention or the functional fragment thereof further inhibitsthe binding of FGFR2 to its ligand.

(3-11) Antibody Binding to Epitope

An “antibody binding to the same site” as in the case of the antibodyprovided by the present invention is also included in the antibody ofthe present invention. The “antibody binding to the same site” as in thecase of a certain antibody means another antibody that binds to a siteon an antigen molecule recognized by the antibody. If a second antibodybinds to a partial peptide or a partial three-dimensional structure onan antigen molecule bound by a first antibody, the first and secondantibodies are determined as binding to the same site. Alternatively,the first and second antibodies are determined as binding to the samesite by confirming that the second antibody competes with the firstantibody for binding to the antigen, i.e., the second antibodyinterferes with the binding of the first antibody to the antigen, evenif the peptide sequence or three-dimensional structure of the specificbinding site is not determined. When the first and second antibodiesbind to the same site and the first antibody has an effectcharacteristic of one aspect of the antibody of the present invention,such as an antitumor activity, the second antibody also has anexceedingly high probability of having the same activity thereas. Thus,if a second anti-FGFR2 antibody binds to a site bound by a firstanti-FGFR2 antibody, the first and second antibodies are determined asbinding to the same site on the FGFR2 protein. Alternatively, the firstand second anti-FGFR2 antibodies are determined as binding to the samesite on the FGFR2 protein by confirming that the second anti-FGFR2antibody competes with the first anti-FGFR2 antibody for binding to theFGFR2 protein.

The present invention also encompasses an antibody binding to a site onthe FGFR2 protein recognized by the monoclonal antibody FR2-10, FR2-13,or FR2-14 of the present invention.

The antibody binding site can be determined by a method well known bythose skilled in the art, such as immunoassay. For example, a series ofpeptides are prepared by appropriately sequentially cleaving the aminoacid sequence of the antigen from its C terminus or N terminus, and thereactivity of the antibody thereto is studied to roughly determine arecognition site. Then, shorter peptides are synthesized, and thereactivity of the antibody to these peptides can be studied to therebydetermine the binding site. The antigen fragment peptides can beprepared using a technique such as gene recombination or peptidesynthesis.

When the antibody binds to or recognizes the partial conformation of theantigen, the binding site for the antibody can be determined byidentifying amino acid residues on the antigen adjacent to the antibodyusing X-ray structural analysis. For example, the antibody or itsfragment and the antigen or its fragment can be bound to each other andcrystallized, followed by structural analysis to identify each aminoacid residue on the antigen having an interaction distance with theantibody. The interaction distance is 8 angstroms or shorter, preferably6 angstroms or shorter, more preferably 4 angstroms or shorter. One ormore such amino acid residues having an interaction distance with theantibody can constitute a site (epitope) on the antigen to which theantibody binds. Two or more such amino acid residues may not be adjacentto each other on the primary sequence.

The Fab fragment of the rat, chimeric, or humanized FR2-14 antibody andthe D2 fragment (peptide consisting of amino acid positions 128 to 249of SEQ ID NO: 70 (FIG. 78)) of human FGFR2 IIIb are bound to each otherand crystallized under conditions involving 1.1 to 2.1 M ammoniumsulfate-0.15 M Tris-HCl buffer solution (pH 6.5 to 8.5) to obtaincrystals in the tetragonal system with a space group of P41212 and unitcells of a=b=60.57 angstroms and c=331.2 angstroms. A molecularreplacement method can be performed using the three-dimensionalstructure coordinates thereof to determine a phase (see Example 15).

The rat, chimeric, or humanized FR2-14 antibody recognizes partialconformation on human FGFR2 IIIb. The epitope for this antibody isconstituted by tyrosine (Tyr) at residue 155, threonine (Thr) at residue157, lysine (Lys) at residue 176, alanine (Ala) at residue 181, glycine(Gly) at residue 182, glycine (Gly) at residue 183, asparagine (Asn) atresidue 184, proline (Pro) at residue 185, methionine (Met) at residue186, threonine (Thr) at residue 188, glutamine (Gln) at residue 200,glutamic acid (Glu) at residue 201, glycine (Gly) at residue 205,glycine (Gly) at residue 206, lysine (Lys) at residue 208, valine (Val)at residue 209, arginine (Arg) at residue 210, asparagine (Asn) atresidue 211, glutamine (Gln) at residue 212, histidine (His) at residue213, tryptophan (Trp) at residue 214, and isoleucine (Ile) at residue217 in the amino acid sequence (SEQ ID NO: 70; FIG. 78) of human FGFR2IIIb or the amino acid sequence (SEQ ID NO: 71; FIG. 79) of human FGFR2IIIc. In other words, this antibody has an interaction distance witheach of tyrosine (Tyr) at residue 155, threonine (Thr) at residue 157,lysine (Lys) at residue 176, alanine (Ala) at residue 181, glycine (Gly)at residue 182, glycine (Gly) at residue 183, asparagine (Asn) atresidue 184, proline (Pro) at residue 185, methionine (Met) at residue186, threonine (Thr) at residue 188, glutamine (Gln) at residue 200,glutamic acid (Glu) at residue 201, glycine (Gly) at residue 205,glycine (Gly) at residue 206, lysine (Lys) at residue 208, valine (Val)at residue 209, arginine (Arg) at residue 210, asparagine (Asn) atresidue 211, glutamine (Gin) at residue 212, histidine (His) at residue213, tryptophan (Trp) at residue 214, and isoleucine (Ile) at residue217 in the amino acid sequence (SEQ ID NO: 70; FIG. 78) of human FGFRIIIb or the amino acid sequence (SEQ ID NO: 71; FIG. 79) of human FGFR2IIIc. The epitope site for this antibody is also found in the amino acidsequence of human FGFR2 IIIc. The antibody of the present invention orthe functional fragment thereof, or a modified form of the antibody orfunctional fragment also encompasses an antibody binding to this epitopeor having an interaction distance with these amino acid residues, afunctional fragment thereof, or a modified form of the antibody orfunctional fragment.

(3-12) Modified Form of Antibody

The present invention provides a modified form of the antibody orfunctional fragment thereof. The modified form of the antibody of thepresent invention or the functional fragment thereof means an antibodyof the present invention or a functional fragment thereof provided withchemical or biological modification. The chemically modified formincludes, for example, a form having an amino acid skeleton conjugatedwith a chemical moiety, and a form having a chemically modified N-linkedor O-linked carbohydrate chain. The biologically modified form includes,for example, a form that has undergone post-translational modification(e.g., N-linked or O-linked glycosylation, N-terminal or C-terminalprocessing, deamidation, isomerization of aspartic acid, or oxidation ofmethionine), and a form containing a methionine residue added to theN-terminus by expression using prokaryotic host cells. Such a modifiedform is also meant to include a form labeled to permit detection orisolation of the antibody or the antigen of the present invention, forexample, an enzyme-labeled form, a fluorescently labeled form, or anaffinity-labeled form. Such a modified form of the antibody of thepresent invention or the functional fragment thereof is useful forimprovement of the stability or blood retention of the original antibodyof the present invention or the original functional fragment thereof,reduction in antigenicity, detection or isolation of the antibody or theantigen, etc.

Examples of the chemical moiety contained in the chemically modifiedform can include water-soluble polymers such as polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, and polyvinyl alcohol.

Examples of the biologically modified form can include a form modifiedby enzymatic treatment, cell treatment, or the like, a form fused withanother peptide, such as a tag, added by gene recombination, and a formprepared from host cells expressing an endogenous or exogenous sugarchain-modifying enzyme.

The antibody dependent cellular cytotoxic activity of the antibody ofthe present invention or the functional fragment thereof may be enhancedby regulating the modification (glycosylation, defucosylation, etc.) ofthe sugar chain bound with the antibody or functional fragment. Forexample, methods described in WO99/54342, WO00/61739, and WO02/31140 areknown as such a technique of regulating the sugar chain modification ofthe antibody, though this technique is not limited thereto. The modifiedform of the antibody of the present invention also includes an antibodythat has undergone the sugar chain modification thus regulated.

Such a modification may be made at an arbitrary position or a desiredposition in the antibody or functional fragment thereof. Alternatively,the same or two or more different modifications may be made at one ortwo or more positions therein.

In the present invention, the “modified form of the antibody fragment”is also meant to include even a “fragment of the modified form of theantibody”.

In the present invention, the modified form of the antibody or themodified form of the functional fragment thereof is also simply referredto as an “antibody” or a “functional fragment of the antibody”.

hFR2-14_H19/L1 is a humanized antibody with regulated sugar chainmodification obtained in Example 9. The nucleotide sequence of the lightchain of this antibody comprises nucleotide positions 61 to 705 of SEQID NO: 72 (FIG. 80), and its amino acid sequence comprises amino acidpositions 21 to 235 of SEQ ID NO: 73 (FIG. 81). The nucleotide sequenceof the heavy chain of this antibody comprises nucleotide positions 58 to1401 of SEQ ID NO: 96 (FIG. 104), and its amino acid sequence comprisesamino acid positions 20 to 467 of SEQ ID NO: 97 (FIG. 105). Such ahumanized antibody is also encompassed by the antibody of the presentinvention or the modified form of the antibody of the present invention.

4. Method for Producing Antibody

(4-1) Method Using Hybridoma

In order to prepare the anti-FGFR2 antibody of the present invention,anti-FGFR2 antibody-producing cells are isolated from the spleens ofanimals immunized with the FGFR2 protein or its soluble form accordingto the method of Kohler and Milstein (Kohler and Milstein, Nature(1975), 256, p. 495-497; and Kennet, R. ed., Monoclonal Antibodies, p.365-367, Plenum Press, N.Y. (1980)). The cells are fused with myelomacells to thereby establish hybridomas. Monoclonal antibodies can beobtained from cultures of these hybridomas.

(4-1-1) Preparation of Antigen

The antigen for the preparation of the anti-FGFR2 antibody can beobtained according to, for example, the method for preparing a native orrecombinant FGFR2 protein described in other paragraphs of the presentspecification. Examples of the antigen that may be thus prepared caninclude the FGFR2 protein and an FGFR2 protein fragment comprising apartial sequence with at least 6 consecutive amino acids of the FGFR2protein, and their derivatives further comprising an arbitrary aminoacid sequence or carrier added thereto (hereinafter, collectivelyreferred to as an “FGFR2 antigen”).

The recombinant FGFR2 antigen can be prepared by transfecting host cellswith a gene comprising a nucleotide sequence encoding the amino acidsequence of the FGFR2 antigen, and recovering the antigen from culturesof the cells. Such a recombinant antigen may be a fusion protein withanother protein such as an immunoglobulin Fc region. An FGFR2 antigenobtained in a cell-free in vitro translation system from a genecomprising a nucleotide sequence encoding the amino acid sequence of theFGFR2 antigen is also included in the recombinant FGFR2 antigen. Thenon-recombinant FGFR2 antigen can be purified or isolated fromFGFR-expressing normal tissues, cancer tissues, or cancer cells,cultures of the cancer cells, or the like described in (iv) of paragraph(2-2).

(4-1-2) Production of Anti-FGFR2 Monoclonal Antibody

The monoclonal antibody is typically produced through the followingsteps:

(a) preparing an antigen,(b) preparing antibody-producing cells,(c) preparing myeloma cells (hereinafter, referred to as “myelomas”),(d) fusing the antibody-producing cells with the myelomas,(e) screening for a hybridoma group producing the antibody of interest,and(f) obtaining single cell clones (cloning).

This production method further involves (g) a step of culturing thehybridomas, a step of raising hybridoma-transplanted animals, etc., and(h) a step of assaying or determining the biological activity of themonoclonal antibody, etc., if necessary.

Hereinafter, the method for preparing the monoclonal antibody will bedescribed in detail with reference to these steps. However, the methodfor preparing the antibody is not limited to those steps, and, forexample, antibody-producing cells other than spleen cells and myelomasmay be used.

(a) Purification of Antigen

This step is performed according to the method for preparing the FGFR2protein described above in (2-3).

(b) Step of Preparing Antibody-Producing Cell

The antigen obtained in step (a) is mixed with an adjuvant such as acomplete or incomplete Freund's adjuvant or potassium aluminum sulfate,and laboratory animals are immunized with the resulting immunogen. Anylaboratory animal used in a hybridoma preparation method known in theart can be used without limitations. Specifically, for example, mice,rats, goats, sheep, cattle, or horses can be used. From the viewpoint ofreadily available myeloma cells to be fused with isolatedantibody-producing cells, etc., the animals to be immunized arepreferably mice or rats.

The strain of mice or rats actually used is not particularly limited. Inthe case of mice, for example, A, AKR, BALB/c, BALB/cAnNCrj, BDP, BA,CE, C3H, 57BL, C57BL, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, R III,SJL, SWR, WB, or 129 can be used. In the case of rats, for example,Wistar, Low, Lewis, Sprague-Dawley, ACI, BN, or Fischer can be used.

These mice and rats are available from laboratory animal breeders ordistributors, for example, CLEA Japan, Inc. or Charles RiverLaboratories Japan Inc.

Of those mice and rats, a BALB/c mouse strain or Wistar and Low ratstrains are particularly preferred as animals to be immunized inconsideration of fusion compatibility with the myeloma cells describedlater.

Also, in consideration of the homology between human and mouse antigens,mice whose biological mechanism to remove autoantibodies has beenreduced, i.e., autoimmune disease mice, are also preferably used.

In this context, these mice or rats are preferably 5 to 12 weeks old,more preferably 6 to 8 weeks old, at the time of immunization.

The animals can be immunized with the FGFR2 protein using, for example,the method of Weir, D. M., Handbook of Experimental Immunology Vol. I.II. III., Blackwell Scientific Publications, Oxford (1987), Kabat, E. A.and Mayer, M. M., Experimental Immunochemistry, Charles C ThomasPublisher Spigfield, Ill. (1964).

Examples of methods for determining antibody titers can include, but arenot limited to, immunoassay such as RIA and ELISA.

Antibody-producing cells derived from spleen cells or lymphocytesseparated from the immunized animals, can be prepared according to amethod known in the art, for example, Kohler et al., Nature (1975) 256,p. 495; Kohler et al., Eur. J. Immnol. (1977) 6, p. 511; Milstein etal., Nature (1977), 266, p. 550; Walsh, Nature, (1977) 266, p. 495.

In the case of spleen cells, a general method can be adopted, whichinvolves chopping the spleens, filtering cells through a stainless mesh,and then floating the resulting cells in an Eagle's minimum essentialmedium (MEM) or the like, to separate antibody-producing cells.

(c) Step of Preparing Myeloma

The myeloma cells used in cell fusion are not particularly limited andcan be selected appropriately for use from cell lines known in the art.For example, a hypoxanthine-guanine phosphoribosyl transferase(HGPRT)-deficient line, i.e., mouse-derived X63-Ag8 (X63), NS1-ANS/1(NS1), P3X63-Ag8.Ul (P3Ul), X63-Ag8.653 (X63.653), SP2/0-Ag14 (SP2/0),MPC11-45.6TG1.7 (45.6TG), FO, S149/5XXO, or BU.1, rat-derived210.RSY3.Ag.1.2.3 (Y3), or human-derived U266AR (SKO-007),GM1500-GTG-A12 (GM1500), UC729-6, LICR-LOW-HMy2 (HMy2), or 8226AR/NIP4-1(NP41), whose screening procedures have already been established, ispreferably used in consideration of convenience in the selection ofhybridomas from fusion cells. These HGPRT-deficient lines are availablefrom, for example, American Type Culture Collection (ATCC).

These cell lines are subcultured in an appropriate medium, for example,an 8-azaguanine medium [RPMI-1640 medium supplemented with glutamine,2-mercaptoethanol, gentamicin, and fetal bovine serum (hereinafter,referred to as “FBS”) and further supplemented with 8-azaguanine], anIscove's modified Dulbecco's medium (hereinafter, referred to as“IMDM”), or a Dulbecco's modified Eagle medium (hereinafter, referred toas “DMEM”) and subcultured in a normal medium [e.g., ASF104 medium(manufactured by Ajinomoto Co., Inc.) containing 10% FBS] 3 to 4 daysbefore cell fusion to secure that the number of cells is equal to orgreater than 2×10⁷ cells on the day of cell fusion.

(d) Step of Fusing Antibody-Producing Cell with Myeloma Cell

The antibody-producing cells can be fused with the myeloma cells underconditions that prevent cell viability from being exceedingly reduced,according to any method known in the art (e.g., Weir, D. M., Handbook ofExperimental Immunology Vol. I. II. III., Blackwell ScientificPublications, Oxford (1987), Kabat, E. A. and Mayer, M. M., ExperimentalImmunochemistry, Charles C Thomas Publisher Spigfield, Ill. (1964)). Forexample, a chemical method which involves mixing antibody-producingcells with myeloma cells in a high-concentration solution of a polymersuch as polyethylene glycol, or a physical method using electricstimulation can be used.

(e) Step of Screening for Hybridoma Group Producing Antibody of Interest

A method for selection from the hybridomas obtained by cell fusion isnot particularly limited, and a hypoxanthine-aminopterin-thymidine (HAT)selection method (Kohler et al., Nature (1975) 256, p. 495; Milstein etal., Nature (1977) 266, p. 550) is typically used. This method iseffective for obtaining hybridomas using an HGPRT-deficient myeloma cellline, which cannot survive in the presence of aminopterin. Specifically,unfused cells and hybridomas can be cultured in a HAT medium to therebyallow only hybridomas resistant to aminopterin to selectively live andgrow.

(f) Step of Obtaining Single Cell Clone (Cloning)

The hybridomas can be cloned using any method known in the art, forexample, a methylcellulose, soft agarose, or limiting dilution method(see e.g., Barbara, B. M. and Stanley, M. S.: Selected Methods inCellular Immunology, W.H. Freeman and Company, San Francisco (1980)).The limiting dilution method is preferred.

(g) Step of Culturing Hybridoma and Step of RaisingHybridoma-Transplanted Animal

The selected hybridomas can be cultured to thereby produce monoclonalantibodies. Preferably, the desired hybridomas are cloned and thensubjected to antibody production.

The monoclonal antibody produced by such a hybridoma can be recoveredfrom cultures of the hybridoma. Also, a recombinant antibody can berecovered from cultures of cells transfected with the monoclonalantibody gene. Alternatively, the hybridoma may be injectedintraperitoneally to mice of the same strain (e.g., BALB/cAnNCrjdescribed above) or Nu/Nu mice and allowed to grow. Then, the monoclonalantibody can be recovered from their ascites.

(h) Step of Assaying or Determining Biological Activity of MonoclonalAntibody

Various biological tests can be selected and applied thereto accordingto the purpose.

(4-2) Cell Immunization Method

Cells expressing the native FGFR2 protein, cells expressing therecombinant FGFR2 protein or its fragment, or the like, can be used asimmunogens to thereby prepare an anti-FGFR2 antibody by the hybridomamethod described above.

Examples of the cells expressing the native FGFR2 protein can includeFGFR2-expressing cells, cell lines derived from FGFR2-expressing tissuesor cancer, and cell lines derived from cancer tissues in which switchingfrom FGFR2 IIIb to FGFR2 IIIc expression is seen. Cancer highlyexpressing FGFR2 includes: cancers found to have gene amplification,such as stomach cancer and breast cancer; and cancers found to haveoverexpression, such as pancreatic cancer and ovary cancer. Examples ofcultured cell lines highly expressing FGFR2 IIIb can include stomachcancer cell lines and breast cancer cell lines. Examples of culturedcell lines highly expressing FGFR2 IIIc can include colorectal (cecal)cancer cell lines. Examples of the cancer tissues in which switchingfrom FGFR2 IIIb to FGFR2 IIIc expression is seen can include tissues ofprostate cancer, urinary bladder cancer, and breast cancer. Examples ofthe cancer tissues expressing FGFR2 IIIc can include tissues of uterinecervix cancer and non-small cell lung cancer. Of these cancers, uterinecervix cancer highly expresses FGFR2 IIIc. Examples of the normaltissues highly expressing FGFR2 can include the brain, the largeintestine, thyroid glands, the uterine, the gallbladder, and the skin.

These FGFR2-expressing cells are used in an amount of 1×10⁵ to 1×10⁹cells, preferably 1×10⁶ to 1×10⁸ cells, more preferably 0.5 to 2×10⁷cells, even more preferably 1×10⁷ cells, per immunization shot. Thenumber of cells used for immunization can be changed according to theexpression level of the FGFR2 protein. The immunogens are generallyadministered intraperitoneally and may be administered through anintradermal route or the like. The hybridomas can be prepared by theapplication of the method described in paragraph (4-1-2).

(4-3) Gene Recombination

In order to prepare the antibody of the present invention, a nucleotide(heavy chain nucleotide) comprising a nucleotide sequence encoding theamino acid sequence of its heavy chain and a nucleotide (light chainnucleotide) comprising a nucleotide sequence encoding the amino acidsequence of its light chain, or a vector having an insert of the heavychain nucleotide and a vector having an insert of the light chainnucleotide are introduced into host cells, and then the cells arecultured, and the antibody can be recovered from the cultures. The heavychain nucleotide and the light chain nucleotide may be inserted in onevector.

Prokaryotic or eukaryotic cells can be used as the host cells. In thecase of using host eukaryotic cells, animal cells, plant cells, oreukaryotic microbes can be used.

Examples of the animal cells can include mammal-derived cells, i.e.,monkey-derived COS cells (Gluzman, Y. Cell (1981), 23, p. 175-182, ATCCCRL-1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658), a mouse NS0 cellline (ECACC), Chinese hamster ovary cells (CHO cells, ATCC CCL-61),dihydrofolate reductase-deficient lines thereof (CHO^(dhfr-); Urlaub, G.and Chasin, L. A. Proc. Natl. Acad. Sci. U.S.A. (1980), 77, p.4126-4220), CHOK1SV (Lonza Biologics), cells derived from birds such aschickens, and cells derived from insects.

Also, cells modified to regulate the sugar chain modification ofproteins such as antibodies can be used as the hosts. For example, CHOcells modified such that sugar chains with fucose bound toN-acetylglucosamine at their reducing ends are reduced or removed amongcomplex-type N-glycoside-linked sugar chains binding to the Fc region ofthe antibody, may be used in antibody expression to thereby prepare adefucosylated antibody (also referred to as a modified form of theantibody) (WO00/61739, WO02/31140, etc.).

Examples of the eukaryotic microbes can include yeasts.

Examples of the prokaryotic cells can include E. coli and Bacillussubtilis.

A signal peptide for the secretion of the antibody of the presentinvention (monoclonal antibody derived from each animal, rat antibody,mouse antibody, chimeric antibody, humanized antibody, human antibody,etc.) is not limited to the secretory signal of an antibody of the samespecies, the same type, and the same subtype as the antibody of thepresent invention or to the antibody of the present invention's ownsecretory signal. Any secretory signal of an antibody of different typeor subtype therefrom or any secretory signal of a protein derived from adifferent eukaryotic species therefrom or a prokaryotic species can beselected and used.

(4-4) Methods for Designing and Preparing Humanized Antibody

Examples of the humanized antibody can include, but are not limited to,a human-derived antibody having CDRs replaced with the CDRs of anon-human animal antibody (see Nature (1986), 321, p. 522-525), a humanantibody grafted with the CDR sequences and with some amino acidresidues of framework regions by CDR grafting (see WO90/07861 and U.S.Pat. No. 6,972,323), and an antibody having human antibody amino acid(s)replaced for one or two or more non-human animal antibody-derived aminoacid(s) in any of these humanized antibodies.

(4-5) Method for Preparing Human Antibody

Further examples of the antibody of the present invention can include ahuman antibody. The anti-FGFR2 human antibody means an anti-FGFR2antibody consisting of the amino acid sequence of a human-derivedantibody. The anti-FGFR2 human antibody can be obtained by a methodusing human antibody-producing mice carrying human genomic DNA fragmentscomprising human antibody heavy and light chain genes (see e.g.,Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133-143; Kuroiwa, Y.et. al., Nuc. Acids Res. (1998) 26, p. 3447-3448; Yoshida, H. et. al.,Animal Cell Technology: Basic and Applied Aspects vol. 10, p. 69-73(Kitagawa, Y., Matuda, T. and Iijima, S. eds.), Kluwer AcademicPublishers, 1999; Tomizuka, K. et. al., Proc. Natl. Acad. Sci. USA(2000) 97, p. 722-727).

Specifically, such human antibody-producing animals may be any ofrecombinant animals that are obtained by disrupting the endogenousimmunoglobulin heavy and light chain gene loci of non-human mammals andinstead introducing thereto human immunoglobulin heavy and light chaingene loci via yeast artificial chromosome (YAC) vectors or the like, andrecombinant animals that are created by crossing these animals.

Alternatively, eukaryotic cells may be transfected with cDNAs encodingthe heavy and light chains, respectively, of such a human antibody,preferably with vectors comprising the cDNAs, by a gene recombinationtechnique. The transfected cells producing a recombinant humanmonoclonal antibody can be cultured. This antibody can be obtained fromthe culture supernatant.

In this context, for example, eukaryotic cells, preferably mammaliancells such as CHO cells, lymphocytes, or myelomas, can be used as thehosts.

Also, a method for obtaining a phage display-derived human antibodyselected from a human antibody library (see e.g., Wormstone, I. M. et.al, Investigative Ophthalmology & Visual Science. (2002) 43 (7), p.2301-2308; Carmen, S. et. al., Briefings in Functional Genomics andProteomics (2002), 1 (2), p. 189-203; Siriwardena, D. et. al.,Opthalmology (2002) 109 (3), p. 427-431) is known.

For example, a phage display method (Nature Biotechnology (2005), 23,(9), p. 1105-1116) can be used, which involves allowing the variableregions of a human antibody to be expressed as a single chain antibody(scFv) on phage surface and selecting a phage binding to the antigen.

The phage selected on the basis of its ability to bind to the antigencan be subjected to gene analysis to thereby determine DNA sequencesencoding the variable regions of the human antibody binding to theantigen.

If the DNA sequence of scFv binding to the antigen is determined, anexpression vector having this sequence can be prepared and introduced toappropriate hosts to allow them to express the human antibody(WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438,WO95/15388, Annu. Rev. Immunol (1994) 12, p. 433-455, NatureBiotechnology (2005) 23 (9), p. 1105-1116).

(4-6) Method for Preparing Functional Fragment of Antibody

The method for preparing a single chain antibody is well known in theart (see e.g., U.S. Pat. Nos. 4,946,778, 5,260,203, 5,091,513, and5,455,030). In this scFv, a heavy chain variable region and a lightchain variable region are linked via a linker that prevents them fromforming a conjugate, preferably a polypeptide linker (Huston, J. S. etal., Proc. Natl. Acad. Sci. U.S.A. (1988), 85, p. 5879-5883). The heavychain variable region and the light chain variable region in scFv may bederived from the same antibody or may be derived from differentantibodies.

For example, an arbitrary single chain peptide consisting of 12 to 19residues is used as the polypeptide linker that links these variableregions.

In order to obtain scFv-encoding DNA, of the sequences of DNA encodingthe heavy chain or heavy chain variable region of the antibody and DNAencoding the light chain or light chain variable region thereof, eachDNA portion encoding the whole or desired amino acid sequence is used asa template and amplified by PCR using a primer pair flanking both endsof the template. Subsequently, DNA encoding the polypeptide linkermoiety is further amplified in combination with a primer pair flankingboth ends of the DNA so that the obtained fragment can be linked at itsends to the heavy and light chain DNAs, respectively.

The scFv-encoding DNA can be used to thereby prepare, according to aroutine method, an expression vector containing the DNA and host cellstransformed with the expression vector. In addition, the host cells canbe cultured, and the scFv can be recovered from the cultures accordingto a routine method.

Also in order to obtain any other functional fragment of the antibody, agene encoding the functional fragment is obtained according to themethod described above and introduced into cells. The functionalfragment of interest can be recovered from cultures of the cells.

The antibody of the present invention may be multimerized to therebyenhance its affinity for the antigen. In this case, antibodies of thesame type may be multimerized, or a plurality of antibodies recognizinga plurality of epitopes, respectively, of the same antigen may bemultimerized. Examples of methods for multimerizing these antibodies caninclude the binding of two scFvs to an IgG CH3 domain, the bindingthereof to streptavidin, and the introduction of a helix-turn-helixmotif.

The antibody of the present invention may be a mixture of plural typesof anti-FGFR2 antibodies differing in amino acid sequence, i.e., apolyclonal antibody. Examples of the polyclonal antibody can include amixture of plural types of antibodies differing in a portion or thewhole of CDRs. Such a polyclonal antibody can be recovered from culturesof mixed-cultured different antibody-producing cells (WO2004/061104).Alternatively, separately prepared antibodies may be mixed. Antiserum,which is one aspect of the polyclonal antibody, can be prepared byimmunizing animals with the desired antigen and recovering serum fromthe animals according to a standard method.

Antibodies conjugated with various molecules such as polyethylene glycol(PEG) can also be used as modified forms of the antibody.

The antibody of the present invention may further be any of conjugatesformed by these antibodies with other drugs (immunoconjugates). Examplesof such an antibody can include the antibody conjugated with aradioactive material or a compound having a pharmacological action(Nature Biotechnology (2005), 23, p. 1137-1146).

(4-7) Purification of Antibody

The obtained antibody can be purified until homogeneous. Usual proteinseparation and purification methods can be used for the separation andpurification of the antibody.

The antibody can be separated and purified by appropriately selected orcombined approach(es), for example, chromatography columns, filters,ultrafiltration, salting out, dialysis, preparative polyacrylamide gelelectrophoresis, and/or isoelectric focusing (Strategies for ProteinPurification and Characterization: A Laboratory Course Manual, Daniel R.Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996);Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold SpringHarbor Laboratory (1988)) though the separation and purification methodis not limited thereto.

Examples of chromatography include affinity chromatography, ion-exchangechromatography, hydrophobic chromatography, gel filtration,reverse-phase chromatography, and adsorption chromatography.

These chromatography approaches can be performed using liquid-phasechromatography such as HPLC or FPLC.

Examples of columns used in affinity chromatography can include proteinA, protein G, and antigen columns.

Examples of the protein A columns include Protein A Ceramic HyperD(manufactured by Pall Corp.), POROS (manufactured by Applied Biosystems,Inc.), and Sepharose F. F. (manufactured by GE Healthcare Bio-SciencesCorp.).

Also, the antibody may be purified using its binding activity againstthe antigen using an antigen-immobilized carrier.

(4-8) Nucleotides Encoding Antibody, Recombinant Vector, and RecombinantCell

The present invention provides a nucleotide(s) encoding the antibody ofthe present invention or the functional fragment thereof, or themodified form of the antibody or functional fragment (hereinafter, thisnucleotide is referred to as an “antibody gene”), a recombinant vectorhaving an insert of the gene, a cell comprising the gene or the vector(hereinafter, this cell is referred to as an “antibody gene-transfectedcell”), and a cell producing the antibody of the present invention orthe functional fragment thereof, or the modified form of the antibody orfunctional fragment (hereinafter, this cell is referred to as an“antibody-producing cell”).

Preferably, the antibody gene of the present invention comprises anucleotide sequence described in any one of the following (a) to (e)(hereinafter, referred to as an “antibody gene sequence”), consists of anucleotide sequence comprising the antibody gene sequence, or consistsof the antibody gene sequence:

(a) a combination of a nucleotide sequence encoding the heavy chainamino acid sequence of any one of the rat FR2-10, FR2-13, and FR2-14,chimeric cFR2-10, cFR2-13, and cFR2-14, and humanized hFR2-14_H1/L1 tohFR2-14_H19/L1 antibodies and a nucleotide sequence encoding the lightchain amino acid sequence of any one thereof;(b) a combination of a nucleotide sequence encoding the amino acidsequence of a heavy chain comprising CDRH1 to CDRH3 of any one of therat FR2-10, FR2-13, and FR2-14, chimeric cFR2-10, cFR2-13, and cFR2-14,and humanized hFR2-14_H1/L1 to hFR2-14_H19/L1 antibodies and anucleotide sequence encoding the amino acid sequence of a light chaincomprising CDRL1 to CDRL3 of any one thereof;(c) a combination of a nucleotide sequence encoding a heavy chain aminoacid sequence comprising the amino acid sequence of the heavy chainvariable region of any one of the rat FR2-10, FR2-13, and FR2-14,chimeric cFR2-10, cFR2-13, and cFR2-14, and humanized hFR2-14_H1/L1 tohFR2-14_H19/L1 antibodies and a nucleotide sequence encoding a lightchain amino acid sequence comprising the amino acid sequence of thelight chain variable region of any one thereof;(d) a nucleotide sequence that hybridizes under stringent conditions toa nucleotide consisting of a nucleotide sequence complementary to anyone of the nucleotide sequences (a) to (c) and encodes the amino acidsequence of an antibody binding to FGFR2; and(e) a nucleotide sequence that encodes an amino acid sequence derivedfrom any one of the amino acid sequences (a) to (c) by the substitution,deletion, addition, or insertion of 1 to 50, 1 to 45, 1 to 40, 1 to 30,1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 or 2, or 1 amino acid(s) and encodes the amino acid sequence of anantibody binding to FGFR2.

The antibody having the amino acid sequence encoded by the nucleotidesequence (d) or (e) may have one or two or more of the activitiesdescribed in paragraphs (3-3) to (3-6), in addition to FGFR2 bindingactivity.

However, the antibody gene of the present invention is not limited tothose described in (a) to (e).

The present invention provides, as described in paragraph (4-3), amethod for producing the antibody of the present invention or thefunctional fragment thereof, or the modified form of the antibody orfunctional fragment, comprising the steps of: culturing the antibodygene-transfected cell of the present invention and recovering theantibody, the functional fragment, or the modified form from thecultures. The antibody or functional fragment thereof, or the modifiedform of the antibody or functional fragment obtained by this productionmethod is also included in the present invention.

5. Pharmaceutical Composition

The present invention provides a pharmaceutical composition comprisingthe anti-FGFR2 antibody or functional fragment thereof, or the modifiedform of the antibody or functional fragment.

The pharmaceutical composition of the present invention is useful in thetreatment or prevention of various diseases that are initiated orexacerbated by abnormal or increased FGFR2 signals due to overexpressionof FGFR2 or its ligand or FGFR2 mutations or gene amplification, or byisoform switching of FGFR2 (hereinafter, these diseases are referred toas “FGFR2-related diseases”), particularly, various cancers.

Examples of causes of the initiation or exacerbation of such cancers tobe treated or prevented can include single nucleotide polymorphism (SNP)in an intron of the FGFR2 gene, high expression of FGFR2, missensemutations that constitutively activate FGFR2, amplification oroverexpression of the FGFR2 gene, and switching from FGFR2 IIIb to FGFR2IIIc.

Examples of such cancer types can include breast cancer, endometrialcancer, ovary cancer, lung cancer (e.g., non-small cell lung cancer),stomach cancer, prostate cancer, kidney cancer, liver cancer, pancreaticcancer, colorectal cancer, esophageal cancer, urinary bladder cancer,uterine cervix cancer, blood cancer, lymphoma, and malignant melanoma.Preferred examples thereof can include these cancers expressing theFGFR2 protein.

In the present invention, the treatment or prevention of a diseaseincludes, but is not limited to, the prevention of the onset of thedisease, preferably the disease in an individual expressing the FGFR2protein, the suppression or inhibition of exacerbation or progressionthereof, the alleviation of one or two or more symptoms exhibited by anindividual affected with the disease, the suppression or remission ofexacerbation or progression thereof, the treatment or prevention of asecondary disease, etc.

The pharmaceutical composition of the present invention can comprise atherapeutically or prophylactically effective amount of the anti-FGFR2antibody or the functional fragment of the antibody and apharmaceutically acceptable diluent, vehicle, solubilizer, emulsifier,preservative, and/or additive.

The “therapeutically or prophylactically effective amount” means anamount that exerts therapeutic or prophylactic effects on a particulardisease by means of a particular dosage form and administration routeand has the same meaning as a “pharmacologically effective amount”.

The pharmaceutical composition of the present invention may comprisematerials for changing, maintaining, or retaining pH, osmotic pressure,viscosity, transparency, color, tonicity, sterility, or the stability,solubility, sustained release, absorbability, permeability, dosage form,strength, properties, shape, etc., of the composition or the antibodycomprised therein (hereinafter, referred to as “pharmaceuticalmaterials”). The pharmaceutical materials are not particularly limitedas long as the materials are pharmacologically acceptable. For example,no or low toxicity is a property preferably possessed by thesepharmaceutical materials.

Examples of the pharmaceutical materials can include, but are notlimited to, the following: amino acids such as glycine, alanine,glutamine, asparagine, histidine, arginine, and lysine; antimicrobialagents; antioxidants such as ascorbic acid, sodium sulfate, and sodiumbisulfite; buffers such as phosphate, citrate, or borate buffers, sodiumbicarbonate, and Tris-HCl solutions; fillers such as mannitol andglycine; chelating agents such as ethylenediaminetetraacetic acid(EDTA); complexing agents such as caffeine, polyvinylpyrrolidine,β-cyclodextrin, and hydroxypropyl-β-cyclodextrin; bulking agents such asglucose, mannose, and dextrin; other hydrocarbons such asmonosaccharides, disaccharides, glucose, mannose, and dextrin; coloringagents; corrigents; diluents; emulsifiers; hydrophilic polymers such aspolyvinylpyrrolidine; low-molecular-weight polypeptides; salt-formingcounterions; antiseptics such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid, and hydrogen peroxide;solvents such as glycerin, propylene glycol, and polyethylene glycol;sugar alcohols such as mannitol and sorbitol; suspending agents;surfactants such as PEG, sorbitan ester, polysorbates such aspolysorbate 20 and polysorbate 80, triton, tromethamine, lecithin, andcholesterol; stability enhancers such as sucrose and sorbitol;elasticity enhancers such as sodium chloride, potassium chloride,mannitol, and sorbitol; transport agents; diluents; excipients; and/orpharmaceutical additives.

The amount of these pharmaceutical materials added is 0.001 to 1000times, preferably 0.01 to 100 times, more preferably 0.1 to 10 times theweight of the anti-FGFR2 antibody or functional fragment thereof, or themodified form of the antibody or functional fragment.

An immunoliposome comprising the anti-FGFR2 antibody or functionalfragment thereof, or the modified form of the antibody or functionalfragment encapsulated in a liposome, or a modified antibody formcomprising the antibody conjugated with a liposome (U.S. Pat. No.6,214,388, etc.) is also included in the pharmaceutical composition ofthe present invention.

The excipients or vehicles are not particularly limited as long as theyare liquid or solid materials usually used in injectable water, saline,artificial cerebrospinal fluids, and other preparations for oral orparenteral administration. Examples of saline can include neutral salineand serum albumin-containing saline.

Examples of buffers can include a Tris buffer adjusted to bring thefinal pH of the pharmaceutical composition to 7.0 to 8.5, an acetatebuffer adjusted to bring the final pH thereof to 4.0 to 5.5, a citratebuffer adjusted to bring the final pH thereof to 5.0 to 8.0, and ahistidine buffer adjusted to bring the final pH thereof to 5.0 to 8.0.

The pharmaceutical composition of the present invention is a solid, aliquid, a suspension, or the like. Another example of the pharmaceuticalcomposition of the present invention can include freeze-driedpreparations. The freeze-dried preparations can be formed using anexcipient such as sucrose.

The administration route of the pharmaceutical composition of thepresent invention may be any of enteral administration, localadministration, and parenteral administration. Examples thereof caninclude intravenous administration, intraarterial administration,intramuscular administration, intradermal administration, hypodermicadministration, intraperitoneal administration, transdermaladministration, intraosseous administration, intraarticularadministration, and the like.

The composition of a pharmaceutical composition can be determinedaccording to the administration method, the binding affinity of theantibody for the FGFR2 protein, etc. The anti-FGFR2 antibody of thepresent invention or the functional fragment thereof, or the modifiedform of the antibody or functional fragment having higher affinity(lower KD value) for the FGFR2 protein can exert its pharmaceuticalefficacy at a lower dose.

The dose of the anti-FGFR2 antibody of the present invention is notlimited as long as the dose is a pharmacologically effective amount. Thedose can be appropriately determined according to the species of anindividual, the type of disease, symptoms, sex, age, pre-existingconditions, the binding affinity of the antibody for the FGFR2 proteinor its biological activity, and other factors. A dose of usually 0.01 to1000 mg/kg, preferably 0.1 to 100 mg/kg, can be administered once everyday to every 180 days or twice or three or more times a day.

Examples of the form of the pharmaceutical composition can includeinjections (including freeze-dried preparations and drops),suppositories, transnasal absorption preparations, transdermalabsorption preparations, sublingual formulations, capsules, tablets,ointments, granules, aerosols, pills, powders, suspensions, emulsions,eye drops, and biological implant formulations.

The pharmaceutical composition comprising the anti-FGFR2 antibody orfunctional fragment thereof, or the modified form of the antibody orfunctional fragment as an active ingredient can be administeredconcurrently with or separately from an additional drug. For example,the pharmaceutical composition comprising the anti-FGFR2 antibody orfunctional fragment thereof as an active ingredient may be administeredafter administration of the additional drug, or the additional drug maybe administered after administration of the pharmaceutical composition.Alternatively, the pharmaceutical composition and the additional drugmay be administered concurrently. Examples of the additional drug caninclude various anticancer agents such as chemotherapeutics andradiation therapy. These use approaches are collectively referred to as“combined use of the additional drug” with the antibody of the presentinvention. The present invention also encompasses a pharmaceuticalcomposition comprising the antibody of the present invention or thefunctional fragment thereof, or the modified form of the antibody orfunctional fragment and further comprising an additional drug.

The present invention provides a method for treating or preventingFGFR-related diseases such as cancer, use of the antibody of the presentinvention for preparing a pharmaceutical composition for treatment orprevention of the diseases, and use of the antibody of the presentinvention for treating or preventing the diseases. The present inventionalso encompasses a kit for treatment or prevention comprising theantibody of the present invention.

6. Composition for Diagnosis

The present invention provides a composition for testing or diagnosis(hereinafter, referred to as a “composition for diagnosis”) comprisingthe anti-FGFR2 antibody of the present invention or the functionalfragment thereof, or the modified form of the antibody or functionalfragment.

The composition for diagnosis of the present invention is useful in thetesting or diagnosis of FGFR2-related diseases such as cancer or ofFGFR2 expression. In the present invention, the testing or the diagnosisincludes, for example, the determination or measurement of a risk ofdeveloping a disease, the determination of the presence or absence of adisease, the measurement of the degree of progression or exacerbation ofa disease, the measurement or determination of the effect of drugtherapy using the pharmaceutical composition comprising the anti-FGFR2antibody or the like, the measurement or determination of the effect oftherapy other than drug therapy, the measurement of a risk of recurrenceof a disease, and the determination of the presence or absence ofrecurrence of a disease. However, the testing or the diagnosis accordingto the present invention is not limited to these, and any approach canbe used.

The composition for diagnosis of the present invention is useful in theidentification of a recipient individual for the antibody of the presentinvention or the functional fragment thereof, or the modified form ofthe antibody or functional fragment, a composition comprising the same,or a pharmaceutical composition comprising the same.

The composition for diagnosis can comprise a pH buffer, anosmoregulator, salts, a stabilizer, an antiseptic, a color developer, asensitizer, an aggregation inhibitor, and the like.

The present invention provides a method for testing or diagnosingFGFR2-related diseases such as cancer, use of the antibody of thepresent invention for preparing a composition for diagnosis of thediseases, and use of the antibody of the present invention for testingor diagnosing the diseases. The present invention also encompasses a kitfor testing or diagnosis comprising the antibody of the presentinvention.

The desirable testing or diagnosis method involving the antibody of thepresent invention is sandwich ELISA. Any usual detection method usingantibodies, such as ELISA, RIA, enzyme-linked immunospot (ELISPOT)assay, dot blotting, Ouchterlony test, or counterimmunoelectrophoresis(CIE), may be used. The antibodies can be labeled by a method usingbiotin or by any other labeling method that can be carried out inbiochemical analysis using a labeling material such as HRP, alkalinephosphatase, or FITC. A chromogenic substrate such as TMB(3,3′,5,5′-tetramethylbenzidine), BCIP (5-bromo-4-chloro-3-indolylphosphate), ρ-NPP (ρ-nitrophenyl phosphate), OPD (o-Phenylenediamine),ABTS (3-Ethylbenzothiazoline-6-sulfonic acid), and SuperSignal ELISAPico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.), afluorescent substrate QuantaBlu™ Fluorogenic Peroxidase Substrate(Thermo Fisher Scientific Inc.), and a chemiluminescent substrate can beused in detection using enzymatic labeling. Samples derived from humansor non-human animals as well as artificially treated samples such asrecombinant proteins can be subjected to this assay. Examples of testsamples derived from individual organisms can include, but are notlimited to, blood, synovial fluids, ascites, lymph, cerebrospinalfluids, tissue homogenate supernatants, and tissue sections.

The sandwich ELISA kit for testing or diagnosis comprising the antibodyof the present invention may comprise a solution of FGFR2 proteinstandards, a coloring reagent, a buffer solution for dilution, anantibody for solid phase, an antibody for detection, and a washingsolution, and the like. Preferably, the amount of the antibody bound tothe antigen can be measured by the application of a method such as anabsorbance, fluorescence, luminescence, or radioisotope (RI) method.Preferably, an absorbance plate reader, a fluorescence plate reader, aluminescence plate reader, an RI liquid scintillation counter, or thelike is used in the measurement.

The present invention provides an antibody useful forimmunohistochemistry (IHC) analysis or a functional fragment thereof,and a modified form of the antibody or functional fragment, and acomposition comprising the same. Such a composition is also encompassedby the “composition for diagnosis” of the present invention.

The immunohistochemistry is not particularly limited as long as thisapproach involves reacting a tissue section with an antigen-bindingantibody (primary antibody) and detecting the primary antibody boundwith the antigen.

Preferably, the tissue section is treated with paraffin. Theparaffin-treated tissue section is deparaffinized, followed by antigenretrieval treatment and nonspecific reaction inhibition treatment.Examples of methods for the antigen retrieval treatment can include heattreatment and enzymatic treatment using trypsin or the like. Heattreatment is preferred. The heat treatment is usually performed underpreferred conditions involving a temperature of 90 to 110° C., pH 8 to10, and a treatment time ranging from 20 to 60 minutes. A Tris-EDTAbuffer solution (e.g., a 10 mM Tris buffer solution containing 1 mMEDTA) or the like can be used in pH adjustment. A method forinactivating an endogenous enzyme having the same or similar catalyticactivity as an enzyme used in color development is usually used as thenonspecific reaction inhibition treatment. For color development throughperoxidase reaction, endogenous peroxidase present in tissues ispreferably inhibited in advance using H₂O₂ or the like. A solvent suchas water or methanol can be used for H₂O₂. The concentration of H₂O₂ is0.1 to 3%, preferably 0.3 to 3%. The H₂O₂ solution can be supplementedwith sodium azide. Also, a blocking method using serum or casein can beused as the nonspecific reaction inhibition treatment. Tissues can betreated with serum or casein before the primary antibody reaction.Alternatively, serum or casein may be contained in a solvent fordiluting the primary antibody.

The reaction conditions for the primary antibody are not particularlylimited and involve a temperature of 20 to 50° C., preferably 25 to 42°C., more preferably 37° C. The reaction time is 5 minutes to all nightand all day, preferably 10 minutes to 6 hours, more preferably 30minutes to 2 hours.

Preferably, an antibody (secondary antibody) capable of being visualizedand binding to the primary antibody can be used in the detection of theprimary antibody. Preferably, the secondary antibody can be visualizedby use of a method involving binding an enzyme such as peroxidase oralkaline phosphatase to the secondary antibody or adding biotin or thelike to the secondary antibody and binding thereto streptavidin or thelike conjugated with the enzyme, followed by reaction with a chromogenicsubstrate compatible with the enzyme. Examples of the method involvingbinding an enzyme to the secondary antibody can include a method using areagent comprising a dextrin polymer or an amino acid polymer to whichmultiple molecules of the enzyme and the secondary antibody are attached(polymer method). A chromogenic substrate such as DAB can be used in themethod involving reacting a biotinylated secondary antibody withperoxidase-labeled streptavidin (LSAB method).

The immunohistochemistry procedure can be performed automatically usingan immunological apparatus programmed with a reaction solution, reactionconditions, the number of washing runs, etc.

The antibody or functional fragment thereof, or the modified form of theantibody or functional fragment comprised in the composition fordiagnosis of the present invention is preferably an antibody binding toFGFR2, i.e., an antibody having FGFR2 selectivity or a functionalfragment thereof, or a modified form of the antibody or functionalfragment, more preferably an antibody having human FGFR2 IIIbselectivity or functional fragment thereof or a modified form of theantibody or functional fragment. More preferably, according to anotheraspect, the antibody or functional fragment thereof, or the modifiedform of the antibody or functional fragment contained in the compositionfor diagnosis of the present invention more has selectivity for bothhuman FGFR2 IIIb and human FGFR2 IIIc.

Examples of the antibody having human FGFR2 IIIb selectivity can includean antibody comprising a heavy chain comprising the heavy chain CDRH1 toCDRH3 of the rat FR2-10 antibody and a light chain comprising the lightchain CDRL1 to CDRL3 thereof, an antibody comprising the heavy and lightchain variable regions of the rat FR2-10 antibody, and an antibodycomprising the heavy and light chains of the rat FR2-10 antibody.Examples of such antibodies can include, but are not limited to, the ratFR2-10 antibody, the chimeric cFR2-10 antibody, and the humanized FR2-10antibodies.

Examples of the antibody having selectivity for both human FGFR2 IIIband human FGFR2 IIIc can include an antibody comprising a heavy chaincomprising the heavy chain CDRH1 to CDRH3 of the rat FR2-13 antibody anda light chain comprising the light chain CDRL1 to CDRL3 thereof, anantibody comprising a heavy chain comprising the heavy chain CDRH1 toCDRH3 of the rat FR2-14 antibody and a light chain comprising the lightchain CDRL1 to CDRL3 thereof, an antibody comprising the heavy and lightchain variable regions of the rat FR2-13 antibody, an antibodycomprising the heavy and light chain variable regions of the rat FR2-14antibody, an antibody comprising the heavy and light chains of the ratFR2-13 antibody, and an antibody comprising the heavy and light chainsof the rat FR2-14 antibody. Examples of such antibodies can include, butare not limited to, the rat FR2-13 antibody, the chimeric cFR2-13antibody, the humanized FR2-13 antibodies, the rat FR2-14 antibody, thechimeric cFR2-14 antibody, and the humanized FR2-14 antibodies.

According to a preferred aspect, the composition for diagnosis of thepresent invention is for detection or assay of FGFR2, more preferablyfor detection or assay of human FGFR2 IIIb and/or human FGFR2 IIIc, evenmore preferably for detection or assay of human FGFR2 IIIb or humanFGFR2 IIIb and human FGFR2 IIIc.

The present invention provides a method for detecting or assaying humanFGFR2 IIIb in a test sample.

Alternatively, human FGFR2 IIIc in a test sample can be detected orassayed by: (i) detecting or assaying human FGFR2 IIIb and human FGFR2IIIc in the test sample; (ii) detecting or assaying human FGFR2 IIIb inthe sample; and (iii) comparing the results of detection or assay in thestep (i) with the results of detection or assay in the step (ii) orsubtracting the results of detection or assay in the step (ii) from theresults of detection or assay in the step (i). Such a method fordetecting or assaying human FGFR2 IIIc is also encompassed in thepresent invention.

The composition for diagnosis of the present invention can be used inthese detection or assay methods. The present invention also encompassessuch an assay method and a composition for diagnosis which are intendedfor diagnosis or testing of human FGFR2-positive cancer, preferably,human FGFR2 IIIb- and/or human FGFR2 IIIc-positive cancer.

The present invention also encompasses a method for identifying arecipient individual for the pharmaceutical composition of the presentinvention. This identification method involves assaying human FGFR2 in asample derived from the individual. The individual can be determined tobe positive when human FGFR2 is detected in the sample or when humanFGFR2 is detected in a larger amount than that of human FGFR2 detectedin a sample derived from a healthy individual. The human FGFR2 in theidentification method is preferably human FGFR2 IIIb and/or human FGFR2IIIc, more preferably human FGFR2 IIIb or human FGFR2 IIIb and humanFGFR2 IIIc.

The composition for diagnosis of the present invention can be used inthis method.

According to a preferred aspect, the individual in the identificationmethod has cancer or is at risk thereof.

According to one aspect, the pharmaceutical composition of the presentinvention can be administered to an individual determined to be positiveby the identification method.

7. Reagent

The antibody of the present invention or the functional fragmentthereof, or the modified form of the antibody or functional fragment isalso useful as a reagent. Such a reagent is used for testing ordiagnosis as mentioned above, for research, and for any other use.

8. Screening Method

The present invention provides a method for identifying a substancehaving FGFR2-neutralizing activity. This method involves identifying asubstance binding to a site on the antigen to which the antibody of thepresent invention binds. For example, a test substance is contacted withthe human FGFR2 IIIb protein or its fragment. Subsequently, the distanceis measured between the substance and amino acid residues constitutingthe epitope on the human FGFR IIIb to which any one of the rat FR2-14antibody, the chimeric cFR2-14 antibody, and the humanized hFR2-14_H1/L1to hFR2-14_H19/L1 antibodies binds. The substance can be determined tobe positive when the substance has an interaction distance with each ofthe residues.

Such an identification method is also useful as a method for identifyinga substance having antitumor activity. The antitumor activity of thesubstance confirmed to be positive by the method may be further assayed.

The test substance is preferably a peptide or an antibody or afunctional fragment thereof, or a modified form of the antibody orfunctional fragment.

The peptide or the antibody or functional fragment thereof, or themodified form of the antibody or functional fragment confirmed to bepositive by the method can also be prepared by gene recombination,peptide synthesis, or in vitro translation. The present invention alsoencompasses a method for producing such a peptide or an antibody, or thelike.

EXAMPLES

Hereinafter, the present invention will be described furtherspecifically with reference to the Examples. However, the presentinvention is not intended to be limited to them.

Procedures related to gene manipulation in the Examples below wereperformed according to the methods described in “Molecular Cloning”(Sambrook, J., Fritsch, E. F. and Maniatis, T., Cold Spring HarborLaboratory Press, 1989) or the methods described in other experimentalmanuals used by those skilled in the art, or using commerciallyavailable reagents or kits according to the instruction manuals, unlessotherwise specified.

Example 1 Preparation of Rat Anti-Human FGFR2 Antibody

1)-1 Immunization

Eight-week-old female WKY/Izm rats (Japan SLC, Inc.) and 7-week-oldfemale Crlj:WI rats (Charles River Laboratories Japan Inc.) were used.At day 0, a mixture of 50 μg of Recombinant Human FGFR2β (IIIb)/FcChimera (manufactured by R&D Systems, Inc.) and Freund's CompleteAdjuvant (manufactured by Wako Pure Chemicals Industries, Ltd.) (volumeratio: 1:2) was administered to the tail base of each WKY/Izm rat. Atday 21, 50 μg of Recombinant Human FGFR2β (IIIb)/Fc Chimera wasadministered to the tail base of each rat. At day 35, the lymph node orthe spleen was collected from the rat and used in hybridoma preparation.At day 0, a mixture of 50 of FGFR2β (IIIb)/Fc Chimera and Freund'sComplete Adjuvant (volume ratio: 1:1) was subcutaneously orintradermally administered to each Crlj:WI rat. At days 7, 14, and 21, amixture of 50 μg of FGFR2β (IIIb)/Fc Chimera and Freund's IncompleteAdjuvant (volume ratio: 1:1) was subcutaneously or intradermallyadministered to the rat. At day 38, 50 μg of FGFR2β (IIIb)/Fc Chimerawas administered into the tail vein of the rat. At day 42, the lymphnode or the spleen was collected from the rat and used in hybridomapreparation.

1)-2 Hybridoma Preparation

The lymph node cells or the spleen cells were electrically fused withmouse myeloma SP2/0-ag14 cells using Hybrimune Hybridoma ProductionSystem (manufactured by Cyto Pulse Sciences, Inc.). The fused cells werediluted with ClonaCell-HY Selection Medium D (manufactured by StemCellTechnologies Inc.) and cultured. Hybridoma colonies that appeared wererecovered to prepare monoclonal hybridomas. Each hybridoma colony thusrecovered was cultured, and the obtained hybridoma culture supernatantwas used to screen for an anti-FGFR2 antibody-producing hybridoma.

1)-3 Construction of Expression Vector for Screening for Antigen-BindingAntibody

1)-3-1 Construction of Human FGFR2 IIIb and FGFR2 IIIc ExpressionVectors (pcDNA-DEST40-FGFR2 IIIb and pcDNA-DEST40-FGFR2 IIIc)

cDNAs encoding a human FGFR2 IIIb variant protein (isoform 2:NP_(—)075259) and a human FGFR2 IIIc variant protein (isoform 1:NP_(—)000132) were cloned into pcDNA-DEST40 vectors to construct vectorspcDNA-DEST40-FGFR2 IIIb and pcDNA-DEST40-FGFR2 IIIc for expression ofeach variant protein, respectively.

1)-3-2 Construction of Ig-Like Domain-Deficient FGFR2 IIIb ExpressionVector

Vectors for expression of a mutant deficient in a region of amino acidpositions 54 to 110 in the full-length amino acid sequence (1 to 822) ofFGFR2 IIIb (hereinafter, referred to as an “IgD1-deletion mutant”), amutant deficient in a region of amino acid positions 154 to 246 therein(hereinafter, referred to as an “IgD2-deletion mutant”), or a mutantdeficient in a region of amino acid positions 263 to 358 therein(hereinafter, referred to as an “IgD3-deletion mutant”) were constructedby PCR with pcDNA-DEST40-FGFR2 IIIb as a template.

1)-4 Antibody Screening by Cell-ELISA

1)-4-1 Preparation of Antigen Gene-Expressing Cell for Cell-ELISA

HEK293 cells were adjusted to 7.5×10⁵ cells/ml in a DMEM mediumcontaining 10% FBS. pcDNA-DEST40-FGFR2 IIIb or a control pcDNA-DEST40was transfected thereto using Lipofectamine 2000 (manufactured by LifeTechnologies Corp.). The resulting cells were dispensed in an amount of50 μl/well to a 96-well half area plate (manufactured by Corning Inc.)and cultured overnight at 37° C. under 5% CO₂ conditions in a DMEMmedium containing 10% FBS. The obtained transfected cells were used inthe attached state in Cell-ELISA.

1)-4-2 Cell-ELISA

After removal of the culture supernatant from the expressionvector-transfected HEK293 cells prepared in Example 1)-4-1, eachhybridoma culture supernatant was added to the pcDNA-DEST40-FGFR2 IIIb-or pcDNA-DEST40-transfected HEK293 cells, and the plate was leftstanding at 4° C. for 1 hour. The cells in the wells were washed oncewith PBS containing 5% FBS. Then, Anti-Rat IgG-Peroxidase antibodyproduced in rabbit (manufactured by Sigma-Aldrich Corp.) diluted500-fold with PBS containing 5% FBS was added thereto, and the plate wasleft standing at 4° C. for 1 hour. The cells in the wells were washed 5times with PBS containing 5% FBS. Then, an OPD chromogenic solution (OPDsolution (o-phenylenediamine dihydrochloride (manufactured by Wako PureChemicals Industries, Ltd.) and H₂O₂ dissolved at concentrations of 0.4mg/ml and 0.6% (v/v), respectively, in 0.05 M trisodium citrate and 0.1M disodium hydrogen phosphate dodecahydrate, pH 4.5)) was added theretoat a concentration of 25 μl/well. Color reaction was performed withoccasional stirring and stopped by the addition of 1 M HCl at aconcentration of 25 μl/well. Then, the absorbance was measured at 490 nmusing a plate reader (ENVISION; PerkinElmer, Inc.). In order to select ahybridoma producing an antibody specifically binding to FGFR2 expressedon cell membrane surface, hybridomas that yielded a culture supernatantexhibiting higher absorbance for the pcDNA-DEST40-FGFR2 IIIb expressionvector-transfected HEK293 cells compared with the controlpcDNA-DEST40-transfected HEK293 cells were selected as anti-FGFR2antibody production-positive hybridomas.

1)-5 Antibody Screening by Flow Cytometry

1)-5-1 Preparation of Antigen Gene-Expressing Cell for Flow CytometryAnalysis

HEK293T cells were inoculated at a density of 5×10⁴ cells/cm² to a225-cm² flask (manufactured by Sumitomo Bakelite Co., Ltd.) and culturedovernight at 37° C. under 5% CO₂ conditions in a DMEM medium containing10% FBS. On the next day, the pcDNA-DEST40-FGFR2 IIIb IgD1-deletionmutant deficient in the N-terminal IgD1 domain or a control pcDNA-DEST40was transfected to the HEK293T cells using Lipofectamine 2000, and thecells were further cultured overnight at 37° C. under 5% CO₂ conditions.On the next day, the expression vector-transfected HEK293T cells weretreated with TrypLE Express (manufactured by Life Technologies Corp.),washed with DMEM containing 10% FBS, and then suspended in PBScontaining 5% FBS. The obtained cell suspension was used in flowcytometry analysis.

1)-5-2 Flow Cytometry Analysis

The FGFR2 IIIb binding specificity of the antibody produced by eachhybridoma determined to be positive by Cell-ELISA in Example 1)-4 wasfurther confirmed by flow cytometry. Each HEK293T cell suspensionprepared in Example 1)-5-1 was centrifuged to remove a supernatant.Then, the pcDNA-DEST40-FGFR2 IIIb IgD1-deletion mutant-transfectedHEK293T cells or the pcDNA-DEST40-transfected HEK293T cells weresuspended by the addition of the hybridoma culture supernatant and leftstanding at 4° C. for 1 hour. The cells were washed twice with PBScontaining 5% FBS, then suspended by the addition of Anti-Rat IgG FITCconjugate (manufactured by Sigma-Aldrich Corp.) diluted 320-fold withPBS containing 5% FBS, and left standing at 4° C. for 1 hour. The cellswere washed 3 times with PBS containing 5% FBS and then resuspended inPBS containing 5% FBS and 2 μg/ml 7-aminoactinomycin D (manufactured byMolecular Probes, Inc.), followed by detection using a flow cytometer(FC500; manufactured by Beckman Coulter Inc.). The data was analyzedusing Flowjo (manufactured by Tree Star Inc.). After removal of7-aminoactinomycin D-positive dead cells by gating, the FITCfluorescence intensity of live cells was plotted to a histogram.Hybridomas that yielded a sample exhibiting a shift to strongerfluorescence intensity in the histogram of the pcDNA-DEST40-FGFR2 IIIbIgD1-deletion mutant-transfected HEK293T cells compared with thefluorescence intensity histogram of the control pcDNA-DEST40-transfectedHEK293T cells were obtained as anti-FGFR2 IIIb antibody-producinghybridomas.

1)-6 Screening Based on Signal-Neutralizing Effect

In order to evaluate the signal-neutralizing effect of the anti-FGFR2antibody produced by each obtained hybridoma, an Elk1 luciferasereporter gene assay system for detecting ERK (extracellularsignal-regulated kinase) activation induced by FGFR2 activation vialigand FGF7 stimulation was constructed by a method shown below and usedin the evaluation of the obtained antibody for its effect.

1)-6-1 Construction of Vector for Reporter Assay

First, a pFR-Luc2CP vector was constructed. pFR-Luc (Stratagene #219005)was cleaved with HindIII, blunt-ended using T4 DNA polymerase, and thencleaved with BamHI to isolate a 140-bp fragment comprising five GAL4binding elements and a TATA box. Next, pGL4.12[luc2CP] (Promega #E6661)was cleaved with EcoICRI and BglII, dephosphorylated, and then ligatedwith the 140-bp fragment to prepare a pFR-Luc2CP vector.

1)-6-2 Elk1 Luciferase Reporter Gene Assay

The Elk1 luciferase reporter gene assay was carried out using eachhybridoma culture supernatant selected in Example 1)-5. A cell line293α, which was HEK293 cells stably transfected with integrin αv andintegrin β3 expression vectors, was transiently cotransfected withpcDNA-DEST40-FGFR2 IIIb or pcDNA-DEST40, pFA2-Elk1 (manufactured byStratagene Corp.), pFR-Luc2CP, and pGL4.74[hRluc/TK] (manufactured byPromega Corp.) according to transfection procedures using Lipofectamine2000 (manufactured by Invitrogen Corp.), inoculated to a white 96-wellcell culture plate (manufactured by Corning Inc.), and culturedovernight at 37° C. under 5% CO₂ conditions. On the next day, theculture supernatant was removed, and the hybridoma culture supernatantdiluted 5-fold with DMEM containing 5% FBS was added at a concentrationof 50 μl/well to the plate. After culture at 37° C. for 1 hour under 5%CO₂ conditions, a ligand human FGF7 (manufactured by R&D systems, Inc.)was added at a final concentration of 10 ng/ml to each well. Afterincubation for 6 hours, cell lysates were prepared and assayed forfirefly luciferase activity (specific signal) and Renilla luciferaseactivity (signal for normalization) using Dual-luciferase reporter assaysystem (manufactured by Promega Corp.). The firefly/Renilla ratio wascalculated to normalize data on each well. Hybridomas FR2-10, FR2-13,and FR2-14 were selected which produced anti-FGFR2 antibodies thatreduced ligand FGF7 dependent reporter activation to a ligand-free levelin the FGFR2-expressing HEK293 reporter cells.

1)-7 Isotyping of Antibody

The anti-FGFR2 antibody-producing hybridomas FR2-10, FR2-13, and FR2-14were isotyped using Rat monoclonal isotyping test kit (manufactured byAbD Serotec). As a result, their isotypes were confirmed to be IgG2a andK chains for FR2-10 and IgG1 and K chains for FR2-13 and FR2-14.

1)-8 Preparation of Monoclonal Antibody

Each monoclonal antibody was purified from the ascites (hereinafter,referred to as an “antibody purification material”) of ahybridoma-transplanted mouse.

The mouse ascites was prepared as follows: first, 7- to 8-week-oldBALB/cAJcl-nu/nu (Japan SLC, Inc.) was treated with pristane(manufactured by Sigma-Aldrich Corp.). Approximately 1 to 4 weeks later,each hybridoma washed with PBS was intraperitoneally transplanted in anamount of 1 to 2×10⁷ cells per mouse. One to 2 weeks later, ascitesaccumulated intraperitoneally was collected, sterilized through a0.22-μm filter, and used as an antibody purification material.

Each antibody was purified using Hitrap MabSelect SuRe (manufactured byGE Healthcare Bio-Sciences Corp.). The antibody purification materialwas applied to a column, which was then washed with PBS, followed byelution with 2 M arginine-HCl (pH 4.0). The eluted antibody solution wasneutralized and then buffer-replaced with PBS. The concentration of thepurified antibody was determined by the elution of the antibody boundwith POROS G 20 μm Column PEEK, 4.6 mm×50 mm, 0.83 ml (manufactured byApplied Biosystems, Inc.) and the subsequent measurement of theabsorbance (O.D. 280 nm) of the eluate. Specifically, the antibodysample diluted with PBS was applied to POROS G 20 μm equilibrated withan equilibration buffer (30.6 mM sodium dihydrogen phosphatedodecahydrate, 19.5 mM monopotassium phosphate, and 0.15 M NaCl, pH7.0). The column was washed with an equilibration buffer, followed bythe elution of the antibody bound with the column using an eluent (0.1%(v/v) HCl and 0.15 M NaCl). The peak area of the absorbance (O.D. 280nm) of the eluate was measured, and the concentration was calculatedaccording to the following expression: Antibody sample concentration(mg/ml)=(Peak area of the antibody sample)/(Peak area of a standard(human IgG1))×Concentration (mg/ml) of the standard×Dilution ratio ofthe sample. Also, the concentration of endotoxin contained in theobtained antibody was measured using Limulus ES-II Single Test Wako(Wako Pure Chemicals Industries, Ltd. 295-51030; containing controlstandard endotoxin) and Toxinometer (Wako Pure Chemicals Industries,Ltd. ET-301 or ET-5000) and confirmed to be 1 EU/mg or lower. Theantibody was used in the subsequent experiments.

Example 2 In Vitro Evaluation of Rat Anti-Human FGFR2 Antibodies(FR2-10, FR2-13, and FR2-14)

2)-1 Study on Selective Binding Activity of Obtained Rat Anti-FGFR2Antibodies (FR2-10, FR2-13, and FR2-14) Against Human FGFR2

2)-1-1 Construction of Human FGFR1 IIIc, Human FGFR3 IIIb, Human FGFR3IIIc, and Human FGFR4 Expression Vectors

cDNAs encoding a human FGFR1 IIIc variant protein (isoform 1:NP_(—)075598), a human FGFR3 IIIb variant protein (isoform 3:NP_(—)001156685), a human FGFR3 IIIc variant protein (isoform 1:NP_(—)000133), and a human FGFR4 protein (isoform 1: NP_(—)002002) werecloned into pcDNA-DEST40 vectors to construct vectors pcDNA-DEST40-FGFR1IIIc, pcDNA-DEST40-FGFR3 IIIb, pcDNA-DEST40-FGFR3 IIIc, andpcDNA-DEST40-FGFR4 for expression of each variant protein, respectively.

2)-1-2 Flow Cytometry Analysis

Each human FGFR expression vector constructed in Examples 1)-3-1 and2)-1-1 was transfected to HEK293T cells by the method shown in Example1)-5-1. The cell suspension was centrifuged to remove the supernatant.Then, these various human FGFR expression vector-transfected HEK293Tcells and the pcDNA-DEST40-transfected HEK293T cells were separatelysuspended by the addition of the hybridoma culture supernatantcontaining the FR2-10, FR2-13, or FR2-14 antibody and left standing at4° C. for 1 hour. The cells were washed twice with PBS containing 5%FBS, then suspended by the addition of Anti-Rat IgG FITC conjugate(manufactured by Sigma-Aldrich Corp.) diluted 320-fold with PBScontaining 5% FBS, and left standing at 4° C. for 1 hour. The cells werewashed 3 times with PBS containing 5% FBS and then resuspended in PBScontaining 5% FBS and 2 μg/ml 7-aminoactinomycin D (manufactured byMolecular Probes, Inc.), followed by detection using a flow cytometer(FC500; manufactured by Beckman Coulter Inc.). The data was analyzedusing Flowjo (manufactured by Tree Star Inc.). After removal of7-aminoactinomycin D-positive dead cells by gating, the FITCfluorescence intensity of live cells was plotted to a histogram tocalculate average fluorescence intensity (MFI). As seen from FIG. 1, therat FR2-10 antibody was shown to selectively bind to human FGFR2 IIIb,while the rat FR2-13 and FR2-14 antibodies were shown to selectivelybind to both human FGFR2 IIIb and FGFR2 IIIc.

2)-2 Identification of Epitope for Obtained Rat Anti-FGFR2 Antibodies(FR2-10, FR2-13, and FR2-14)

Epitopes bound by the obtained rat anti-FGFR2 antibodies were identifiedusing vectors for expression of mutants lacking any one of the threeIg-like domains present in the FGFR2 extracellular region. The FGFR2IIIb IgD1, IgD2, or IgD3-deletion mutant expression vector constructedin Example 1)-3-2 was transfected to HEK293T cells by the method shownin Example 1)-5-1. The cell suspension was centrifuged to remove asupernatant. Then, these various Ig-like domain-deficient FGFR2 IIIbexpression vector-transfected HEK293T cells and thepcDNA-DEST40-transfected HEK293T cells were separately suspended by theaddition of the hybridoma culture supernatant containing the FR2-10,FR2-13, or FR2-14 antibody and left standing at 4° C. for 1 hour. Thecells were washed twice with PBS containing 5% FBS, then suspended bythe addition of Anti-Rat IgG FITC conjugate (manufactured bySigma-Aldrich Corp.) diluted 320-fold with PBS containing 5% FBS, andleft standing at 4° C. for 1 hour. The cells were washed 3 times withPBS containing 5% FBS and then resuspended in PBS containing 5% FBS and2 μg/ml 7-aminoactinomycin D (manufactured by Molecular Probes, Inc.),followed by detection using a flow cytometer (FC500; manufactured byBeckman Coulter Inc.). The data was analyzed using Flowjo (manufacturedby Tree Star Inc.). After removal of 7-aminoactinomycin D-positive deadcells by gating, the FITC fluorescence intensity of live cells wasplotted to a histogram to calculate average fluorescence intensity(MFI). As seen from FIG. 2, the rat FR2-10 antibody was shown to bind tothe Ig-like domain 3 of human FGFR2 IIIb. By contrast, the rat FR2-13and FR2-14 antibodies, which also exhibited binding activity againsthuman FGFR2 IIIc, were shown to bind to the common Ig-like domain 2 ofhuman FGFR2 IIIb and FGFR2 IIIc.

2)-3 Signal-Neutralizing Effects of Obtained Rat Anti-FGFR2 Antibodies(FR2-10, FR2-13, and FR2-14)

In order to evaluate the signal-neutralizing effects of the obtainedantibodies by the Elk1 luciferase reporter gene assay, 293α cells werecotransfected with pcDNA-DEST40-FGFR2 IIIb or pcDNA-DEST40-FGFR2 IIIcconstructed in Example 1)-3-1, pFA2-Elk1 (manufactured by StratageneCorp.), pFR-Luc2CP, and pGL4.74[hRluc/TK] (manufactured by PromegaCorp.) by the method shown in Example 1)-6-2, and cultured overnight at37° C. under 5% CO₂ conditions. On the next day, the culture supernatantwas removed, and the cells were then preincubated for 1 hour with therat FR2-10, FR2-13, or FR2-14 antibody diluted with DMEM containing 2%FBS. Subsequently, a ligand human FGF7 (manufactured by R&D systems,Inc.) or human FGF9 (manufactured by PeproTech Inc.) was added at afinal concentration of 10 ng/ml to each well. After incubation for 6hours, firefly luciferase activity (specific signal) and Renillaluciferase activity (signal for normalization) were assayed usingDual-Glo Luciferase Assay System (manufactured by Promega Corp.). Thefirefly/Renilla ratio was calculated to normalize data on each well. Asshown in FIG. 3A, the rat FR2-10, FR2-13, and FR2-14 antibodiesinhibited ligand FGF7 dependent reporter activation in the FGFR2IIIb-expressing cells. As shown in FIG. 3B, the rat FR2-13 and FR2-14antibodies inhibited ligand FGF9 dependent reporter activity in theFGFR2 IIIc-expressing cells. These results demonstrated that theseantibodies have the effect of inhibiting the activation of FGFR2 by itsligand.

2)-4 FGFR2 Signal Inhibitory Effect of Obtained Rat Anti-FGFR2 Antibody(FR2-10) on Human Cancer Cell Line

The FGFR2 signal inhibitory effect of the obtained antibody was testedusing a stomach cancer cell line SNU-16 endogenously expressing FGFR2.SNU-16 cells (3×10⁶) suspended in an RPMI medium containing 0.1% bovineserum albumin were seeded onto a 12-well plate and incubated overnight.The rat FR2-10 antibody was added thereto, and the cells were incubatedat 37° C. for 1 hour. Then, 10 ng/ml FGF7 (manufactured by R&D systems,Inc.) was added thereto, and the cells were further incubated for 10minutes. Subsequently, the cells were lysed with an RIPA buffer (1%NP-40, 0.5% sodium deoxycholate, and 0.1% SDS in PBS) containingComplete Mini (manufactured by Roche Applied Science) and a phosphataseinhibitor (manufactured by Nacalai Tesque Inc.). The lysates werecentrifuged to obtain the cell lysis solution, and the proteinconcentration was determined using BCA protein assay (manufactured byPierce Biotechnology, Inc.). The lysates were resuspended in aDTT-containing buffer and denatured at 99° C. for 5 minutes. The protein(20 μg/lane) was separated by SDS-PAGE on a 5 to 20% gel. The proteinwas blotted onto a PVDF membrane (manufactured by Bio-Rad Laboratories,Inc.). The membrane was blocked with TBS-T (2 mM Tris, 50 mM NaCl, and0.1% Tween-20 (pH 7.4)) containing 5% skimmed milk (MEGMILK SNOW BRANDCo., Ltd.) at room temperature for 1 hour. Then, antibodies againstFGFR2, phosphorylated FGFR2 (p-FGFR2), FRS2, phosphorylated FRS2(p-FRS2), ERK, or phosphorylated ERK (p-ERK) were added thereto,followed by incubation overnight at 4° C. After washing, the membranewas incubated with a horseradish peroxidase-conjugated anti-rabbitsecondary antibody (Amersham Biosciences Corp.). Immunoreactive bandswere visualized with X-ray films using ECL plus substrate (GE HealthcareBio-Sciences Corp.). As shown in FIG. 4, the ligand FGF7 stimulationincreased the phosphorylation of FGFR2, FRS2, and ERK, whereas the ratFR2-10 antibody inhibited the increase in the phosphorylation of thesemolecules in a concentration dependent manner.

Example 3 Sequencing of cDNAs Encoding Variable Regions of RatAnti-Human FGFR2 Antibodies (FR2-10, FR2-13, and FR2-14)

3)-1 Identification of N-Terminal Amino Acid Sequences of Heavy andLight Chains of Rat FR2-10, FR2-13, and FR2-14 Antibodies

In order to identify the N-terminal amino acid sequences of the heavyand light chains of the rat FR2-10, FR2-13, and FR2-14 antibodies, therat FR2-10, FR2-13, and FR2-14 antibodies purified in Example 1)-8 wereeach separated by SDS-PAGE. After the separation, each protein in thegel was transferred from the gel to Sequi-Blot PVDF membrane (Bio-RadLaboratories, Inc.). The membrane was washed with a washing buffer (25mM NaCl and 10 mM sodium borate buffer, pH 8.0), then stained by dippingin a staining solution (50% methanol, 20% acetic acid, and 0.05%Coomassie brilliant blue) for 5 minutes, and then decolorized with 90%methanol. Bands corresponding to the heavy chain (band with a smallermobility) and light chain (band with a larger mobility) of each antibodyvisualized on the PVDF membrane were excised, and their respectiveN-terminal amino acid sequences were identified by the automatic Edmanmethod (see Edman et al. (1967) Eur. J. Biochem. 1, 80) using ProcisecLC protein sequencer Model 492cLC (Applied Biosystems, Inc.). As aresult, the N-terminal amino acid sequence of the band corresponding tothe heavy chain of FR2-10 was EVQLVESGGGLV (SEQ ID NO: 1 of the SequenceListing; FIG. 9), and the N-terminal amino acid sequence of the bandcorresponding to the light chain thereof was DIQMTQSPSSLSA (SEQ ID NO: 2of the Sequence Listing; FIG. 10).

The N-terminal amino acid sequence of the band corresponding to theheavy chain of FR2-13 was QVKLL (SEQ ID NO: 3 of the Sequence Listing;FIG. 11), and the N-terminal amino acid sequence of the bandcorresponding to the light chain thereof was DIQMTQSPASLSASLGE (SEQ IDNO: 4 of the Sequence Listing; FIG. 12).

The N-terminal amino acid sequence of the band corresponding to theheavy chain of FR2-14 was QVKLL (SEQ ID NO: 5 of the Sequence Listing;FIG. 13), and the N-terminal amino acid sequence of the bandcorresponding to the light chain thereof was DIQMTQSPASLSASLGE (SEQ IDNO: 6 of the Sequence Listing; FIG. 14).

3)-2 Preparation of mRNA from FR2-10-, FR2-13-, and FR2-14-ProducingHybridomas

In order to amplify cDNAs encoding the variable regions of FR2-10,FR2-13, and FR2-14, mRNAs were prepared from each of the FR2-10-,FR2-13-, and FR2-14-producing hybridoma cells using mRNA Isolation kit(Roche Applied Science).

3)-3 Synthesis of cDNA (5′-RACE-Ready cDNA)

cDNAs (5′-RACE-Ready cDNAs) were synthesized using 70 ng of each mRNAprepared in Example 3)-2, PrimeScript Reverse Transcriptase (Takara BioInc.), and SMART RACE cDNA Amplification Kit (Clontech Laboratories,Inc.).

3)-4 5′-RACE PCR Amplification and Sequencing of cDNAs Encoding FR2-10,FR2-13, and FR2-14 Heavy Chain Variable Regions

Since the isotype of the FR2-10 heavy chain was IgG2a and the isotypesof the FR2-13 and FR2-14 heavy chains were IgG1 (Example 1)-7), theprimers used for PCR amplification of the variable region-encoding cDNAof each heavy chain gene were oligonucleotides having the nucleotidesequences of UPM (Universal Primer A Mix; attached to SMART RACE cDNAAmplification Kit) and 5′-GAGTTACTTTTGAGAGCAGTTCCAGGAG-3′ (RG1R1: SEQ IDNO: 7 of the Sequence Listing; FIG. 15). The UPM used was attached toSMART RACE cDNA Amplification Kit (Clontech Laboratories, Inc.), whileRG1R1 was designed from the sequences of rat heavy chain (IgG2a andIgG1) constant regions registered in the database.

cDNAs encoding the heavy chain variable regions of FR2-10, FR2-13, andFR2-14 were each amplified by 5′-RACE PCR using this primer set and thecDNAs (5′-RACE-Ready cDNAs) synthesized in Example 3)-3 as templates.This PCR was carried out on the Touchdown PCR program according to themanual of SMART RACE cDNA Amplification Kit (Clontech Laboratories,Inc.) using polymerase KOD-Plus- (Toyobo Co., Ltd.).

Each heavy chain variable region-encoding cDNA amplified by 5′-RACE PCRwas purified using MinElute PCR Purification Kit (Qiagen N.V.) and thenanalyzed by sequencing.

The sequencing primer used was an oligonucleotide having the nucleotidesequence 5′-GAGTTACTTTTGAGAGCAGTTCCAGGAG-3′ (RG1R1: SEQ ID NO: 7 of theSequence Listing; FIG. 15).

On the basis of the results of this sequencing analysis, a sequencingprimer for a complementary strand of each cDNA was further designed asshown below and used in sequencing analysis.

Sequencing primer for FR2-10 5′-GGTTCTCCCACTCAGTAATC-3′ (10HF: SEQ IDNO: 8 of the Sequence Listing; FIG. 16)

Sequencing primer for FR2-13 5′-CATATGATCAGTGTCCTCTC-3′ (13HF: SEQ IDNO: 9 of the Sequence Listing; FIG. 17)

Sequencing primer for FR2-14 5′-ATATGATCAGTGTCCTCTCC-3′ (14HF: SEQ IDNO: 10 of the Sequence Listing; FIG. 18)

The sequencing analysis was carried out using a gene sequence analyzer(“ABI PRISM 3700 DNA Analyzer; Applied Biosystems, Inc.” or “AppliedBiosystems 3730xl Analyzer; Applied Biosystems, Inc.”). GeneAmp 9700(Applied Biosystems, Inc.) was used in sequencing reaction.

The determined nucleotide sequences of the cDNAs encoding the heavychain variable regions of FR2-10, FR2-13, and FR2-14 and the amino acidsequences of these variable regions are shown in SEQ ID NOs: 11, 13, and15 and SEQ ID NOs: 12, 14, and 16 (FIGS. 19, 21, and 23 and FIGS. 20,22, and 24), respectively, of the Sequence Listing.

The amino acid sequences of the FR2-10, FR2-13, and FR2-14 heavy chainvariable regions determined on the basis of their nucleotide sequenceswere consistent with the N-terminal amino acid sequences determined inExample 3)-1.

3)-5 5′-RACE PCR amplification and sequencing of cDNA encoding FR2-10light chain variable region

Since the isotype of the FR2-10 light chain was κ (Example 1)-7), theprimers used for PCR amplification of the variable region-encoding cDNAof the light chain gene were oligonucleotides having the nucleotidesequences of UPM (Universal Primer A Mix; attached to SMART RACE cDNAAmplification Kit) and 5′-TTCATGAGGCACACGACTGAGGCACCTCC-3′ (RKR3: SEQ IDNO: 17 of the Sequence Listing; FIG. 25). The UPM used was attached toSMART RACE cDNA Amplification Kit (Clontech Laboratories, Inc.), whileRKR3 was designed from the sequences of rat light chain constant regionsregistered in the database.

A cDNA encoding the light chain variable region of FR2-10 was amplifiedby 5′-RACE PCR using this primer set and the cDNA (5′-RACE-Ready cDNA)synthesized in Example 3)-3 as a template. This PCR was carried out onthe Touchdown PCR program according to the manual of SMART RACE cDNAAmplification Kit (Clontech Laboratories, Inc.) using polymeraseKOD-Plus- (Toyobo Co., Ltd.). The light chain variable region-encodingcDNA amplified by 5′-RACE PCR was purified using MinElute PCRPurification Kit (Qiagen N.V.) and then analyzed by sequencing.

The sequencing primer used was an oligonucleotide having the nucleotidesequence 5′-TCCAGTTGCTAACTGTTCCG-3′ (sqRK: SEQ ID NO: 18 of the SequenceListing; FIG. 26) designed from the sequences of rat light chainconstant regions registered in the database.

On the basis of the results of this sequencing analysis, a sequencingprimer for a complementary strand of the cDNA was further designed asshown below and used in sequencing analysis.

Sequencing primer for FR2-10 5′-CAGTGGTATCAACGCAGAG-3′ (10LF: SEQ ID NO:19 of the Sequence Listing; FIG. 27)

Sequencing analysis and sequencing reaction were performed as mentionedabove.

The determined nucleotide sequence of the cDNA encoding the light chainvariable region of FR2-10 and the amino acid sequence of this variableregion are shown in SEQ ID NO: 20 and SEQ ID NO: 21 (FIGS. 28 and 29),respectively, of the Sequence Listing.

The amino acid sequence of the FR2-10 light chain variable regiondetermined on the basis of its nucleotide sequence was consistent withthe N-terminal amino acid sequence determined in Example 3)-1.

3)-6 5′-RACE PCR Amplification and Sequencing of cDNAs Encoding FR2-13and FR2-14 Light Chain Variable Regions

Since the isotypes of the FR2-13 and FR2-14 light chains were κ (Example1)-7), the primers used for PCR amplification of the variableregion-encoding cDNA of each light chain gene were oligonucleotideshaving the nucleotide sequences of UPM (Universal Primer A Mix; attachedto SMART RACE cDNA Amplification Kit) and5′-TACGTGCTGTCTTTGCTGTCCTGATCAG-3′ (RKR6: SEQ ID NO: 22 of the SequenceListing; FIG. 30). The UPM used was attached to SMART RACE cDNAAmplification Kit (Clontech Laboratories, Inc.), while RKR6 was designedfrom the sequences of rat light chain constant regions registered in thedatabase.

Variable region-encoding cDNAs of the light chain genes of FR2-13 andFR2-14 were each amplified by 5′-RACE PCR using this primer set and thecDNAs (5′-RACE-Ready cDNAs) synthesized in Example 3)-3 as templates.This PCR was carried out on the Touchdown PCR program according to themanual of SMART RACE cDNA Amplification Kit (Clontech Laboratories,Inc.) using polymerase KOD-Plus- (Toyobo Co., Ltd.).

Each light chain variable region-encoding cDNA amplified by 5′-RACE PCRwas purified using MinElute PCR Purification Kit (Qiagen N.V.) and thencloned using Zero Blunt TOPO PCR Cloning Kit (Invitrogen Corp.). Thecloned cDNAs encoding the light chain variable regions were analyzed bysequencing.

The sequencing primers used were an oligonucleotide having thenucleotide sequence 5′-TTCATGAGGCACACGACTGAGGCACCTCC-3′ (RKR3: SEQ IDNO: 17 of the Sequence Listing; FIG. 25) designed from the sequences ofrat light chain constant regions registered in the database, and NUP(Nested Universal Primer A: attached to SMART RACE cDNA AmplificationKit).

Sequencing analysis and sequencing reaction were performed as mentionedabove.

The determined nucleotide sequences of the cDNAs encoding the lightchain variable regions of FR2-13 and FR2-14 and the amino acid sequencesof these variable regions are shown in SEQ ID NOs: 23 and 25 (FIGS. 31and 33) and SEQ ID NOs: 24 and 26 (FIGS. 32 and 34), respectively, ofthe Sequence Listing.

The amino acid sequences of the FR2-13 and FR2-14 light chain variableregions determined on the basis of their nucleotide sequences wereconsistent with the N-terminal amino acid sequences determined inExample 3)-1.

Example 4 Preparation of Human Chimeric Anti-FGFR2 Antibodies (cFR2-10,cFR2-13, and cFR2-14)

4)-1 Construction of Chimeric and Humanized Light Chain ExpressionVector pCMA-LK

A plasmid pcDNA3.3-TOPO/LacZ (Invitrogen Corp.) was digested withrestriction enzymes XbaI and PmeI. The obtained fragment ofapproximately 5.4 kb was ligated with a DNA fragment comprising a DNAsequence (shown in SEQ ID NO: 27 (FIG. 35) of the Sequence Listing)encoding a human κ chain secretory signal and a human κ chain constantregion using In-Fusion Advantage PCR cloning kit (Clontech Laboratories,Inc.) to prepare pcDNA3.3/LK.

PCR was performed with pcDNA3.3/LK as a template using a primer setshown below. The obtained fragment of approximately 3.8 kb wasphosphorylated and then self-ligated to construct a chimeric andhumanized light chain expression vector pCMA-LK having a signalsequence, a cloning site, and the nucleotide sequence encoding the humanκ chain constant region, downstream of the CMV promoter.

Primer Set

(3.3-F1: SEQ ID NO: 28 of the Sequence Listing; FIG. 36)5′-tataccgtcgacctctagctagagcttggc-3′(3.3-R1: SEQ ID NO: 29 of the Sequence Listing; FIG. 37)5′-gctatggcagggcctgccgccccgacgttg-3′

4)-2 Construction of Chimeric and Humanized IgG1 Type Heavy ChainExpression Vector pCMA-G1

pCMA-LK was digested with XbaI and PmeI. The obtained DNA fragmentexcept for the DNA sequence encoding the κ chain secretory signal andthe human κ chain constant region was ligated with a DNA fragmentcomprising a DNA sequence (shown in SEQ ID NO: 30 (FIG. 38) of theSequence Listing) encoding the amino acids of a human heavy chain signalsequence and a human IgG1 constant region using In-Fusion Advantage PCRcloning kit (Clontech Laboratories, Inc.) to construct a chimeric andhumanized IgG1 type heavy chain expression vector pCMA-G1 having asignal sequence, a cloning site, and the nucleotide sequence encodingthe human IgG1 heavy chain constant region, downstream of the CMVpromoter.

4)-3 Construction of Human Chimeric FR2-10 Light Chain Expression Vector

A DNA fragment comprising a light chain variable region-encoding cDNAwas amplified using the FR2-10 light chain variable region-encoding cDNAobtained in Example 3) as a template, KOD-Plus- (Toyobo Co., Ltd.), anda primer set shown below, and inserted to the restriction enzymeBsiWI-cleaved site of the general-purpose vector pCMA-LK for chimericand humanized antibody light chain expression using In-Fusion AdvantagePCR cloning kit (Clontech Laboratories, Inc.) to construct a humanchimeric FR2-10 light chain expression vector. The obtained expressionvector was designated as “pCMA-LK/cFR2-10”. The nucleotide sequence ofthe human chimeric FR2-10 light chain and the amino acid sequence ofthis light chain are shown in SEQ ID NOs: 31 and 32 (FIGS. 39 and 40),respectively, of the Sequence Listing.

Primer set for human chimeric FR2-10 light chain5′-atctccggcgcgtacggcgacatccagatgacccagtctccatcttcc-3′ (c10-LF: SEQ IDNO: 33 of the Sequence Listing; FIG. 41)5′-ggagggggcggccacagcccgttttatttccaacttcgtccctg-3′ (c10-LR: SEQ ID NO:34 of the Sequence Listing; FIG. 42)

4)-4 Construction of Human Chimeric FR2-10 Heavy Chain Expression Vector

A DNA fragment comprising a heavy chain variable region-encoding cDNAwas amplified using the FR2-10 heavy chain variable region-encoding cDNAobtained in Example 3) as a template, KOD-Plus- (Toyobo Co., Ltd.), anda primer set shown below, and inserted to the restriction enzymeBlpI-cleaved site of the chimeric and humanized IgG1 type heavy chainexpression vector pCMA-G1 using In-Fusion Advantage PCR cloning kit(Clontech Laboratories, Inc.) to construct a human chimeric FR2-10 heavychain expression vector. The obtained expression vector was designatedas “pCMA-G1/cFR2-10”. The nucleotide sequence encoding the humanchimeric FR2-10 heavy chain and the amino acid sequence of this heavychain are shown in SEQ ID NOs: 35 and 36 (FIGS. 43 and 44),respectively, of the Sequence Listing.

Primer set for human chimeric FR2-10 heavy chain5′-ccagatgggtgctgagcgaggtgcagctggtggagtctgggggaggc-3′ (c10-HF: SEQ IDNO: 37 of the Sequence Listing; FIG. 45)5′-cttggtggaggctgagctgacagtgactgaagttccttgaccccaggc-3′ (c10-HR: SEQ IDNO: 38 of the Sequence Listing; FIG. 46)

4)-5 Construction of Human Chimeric FR2-13 Light Chain Expression Vector

A DNA fragment comprising a light chain variable region-encoding cDNAwas amplified using the FR2-13 light chain variable region-encoding cDNAobtained in Example 3) as a template, KOD-Plus- (Toyobo Co., Ltd.), anda primer set shown below, and inserted to the restriction enzymeBsiWI-cleaved site of the general-purpose vector pCMA-LK for chimericand humanized antibody light chain expression using In-Fusion AdvantagePCR cloning kit (Clontech Laboratories, Inc.) to construct a humanchimeric FR2-13 light chain expression vector. The obtained expressionvector was designated as “pCMA-LK/cFR2-13”. The nucleotide sequenceencoding the human chimeric FR2-13 light chain and the amino acidsequence of this light chain are shown in SEQ ID NOs: 39 and 40 (FIGS.47 and 48), respectively, of the Sequence Listing.

Primer set for human chimeric FR2-13 light chain5′-atctccggcgcgtacggcgacatccagatgacacagtctccagcttcc-3′ (c13-LF: SEQ IDNO: 41 of the Sequence Listing; FIG. 49)5′-ggagggggcggccacagcccgtttcagttccagcttggtcccaac-3′ (c13-LR: SEQ ID NO:42 of the Sequence Listing; FIG. 50)

4)-6 Construction of Human Chimeric FR2-13 Heavy Chain Expression Vector

A DNA fragment comprising a heavy chain variable region-encoding cDNAwas amplified using the FR2-13 heavy chain variable region-encoding cDNAobtained in Example 3) as a template, KOD-Plus- (Toyobo Co., Ltd.), anda primer set shown below, and inserted to the restriction enzymeBlpI-cleaved site of the chimeric and humanized IgG1 type heavy chainexpression vector pCMA-G1 using In-Fusion Advantage PCR cloning kit(Clontech Laboratories, Inc.) to construct a human chimeric FR2-13 heavychain expression vector. The obtained expression vector was designatedas “pCMA-G1/cFR2-13”. The nucleotide sequence encoding the humanchimeric FR2-13 heavy chain and the amino acid sequence of this heavychain are shown in SEQ ID NOs: 43 and 44 (FIGS. 51 and 52),respectively, of the Sequence Listing.

Primer set for human chimeric FR2-13 heavy chain5′-ccagatgggtgctgagccaggttaagctgctgcagtctggggctgag-3′ (c13-HF: SEQ IDNO: 45 of the Sequence Listing; FIG. 53)5′-cttggtggaggctgagctgacagtgaccagagtgccttggccccag-3′ (c13-HR: SEQ ID NO:46 of the Sequence Listing; FIG. 54)

4)-7 Construction of Human Chimeric FR2-14 Light Chain Expression Vector

A DNA fragment comprising a light chain variable region-encoding cDNAwas amplified using the FR2-14 light chain variable region-encoding cDNAobtained in Example 3) as a template, KOD-Plus- (Toyobo Co., Ltd.), anda primer set shown below, and inserted to the restriction enzymeBsiWI-cleaved site of the general-purpose vector pCMA-LK for chimericand humanized antibody light chain expression using In-Fusion AdvantagePCR cloning kit (Clontech Laboratories, Inc.) to construct a humanchimeric FR2-14 light chain expression vector. The obtained expressionvector was designated as “pCMA-LK/cFR2-14”. The nucleotide sequenceencoding the human chimeric FR2-14 light chain and the amino acidsequence of this light chain are shown in SEQ ID NOs: 47 and 48 (FIGS.55 and 56), respectively, of the Sequence Listing.

Primer set for human chimeric FR2-14 light chain5′-atctccggcgcgtacggcgacatccagatgacacagtctccagcttcc-3′ (c13-LF: SEQ IDNO: 41 of the Sequence Listing; FIG. 49)5′-ggagggggcggccacagcccgtttcagttccagcttggtcccagc-3′ (c14-LR: SEQ ID NO:49 of the Sequence Listing; FIG. 57)

4)-8 Construction of Human Chimeric FR2-14 Heavy Chain Expression Vector

A DNA fragment comprising a heavy chain variable region-encoding cDNAwas amplified using the FR2-14 heavy chain variable region-encoding cDNAobtained in Example 3) as a template, KOD-Plus- (Toyobo Co., Ltd.), anda primer set shown below, and inserted to the restriction enzymeBlpI-cleaved site of the chimeric and humanized IgG1 type heavy chainexpression vector pCMA-G1 using In-Fusion Advantage PCR cloning kit(Clontech Laboratories, Inc.) to construct a human chimeric FR2-14 heavychain expression vector. The obtained expression vector was designatedas “pCMA-G1/cFR2-14”. The nucleotide sequence encoding the humanchimeric FR2-14 heavy chain and the amino acid sequence of this heavychain are shown in SEQ ID NOs: 50 and 51 (FIGS. 58 and 59),respectively, of the Sequence Listing.

Primer set for human chimeric FR2-14 heavy chain5′-ccagatgggtgctgagccaggttaagctgctgcagtctggggctgag-3′ (c13-HF: SEQ IDNO: 45 of the Sequence Listing; FIG. 53)5′-cttggtggaggctgagctgacagtgaccagagtgccttggccccag-3′ (c13-HR: SEQ ID NO:46 of the Sequence Listing; FIG. 54)

4)-9 Preparation of Human Chimeric Anti-FGFR2 Antibody

4)-9-1 Production of Human Chimeric Anti-FGFR2 Antibody

FreeStyle 293F cells (Invitrogen Corp.) were subcultured and culturedaccording to the manual.

1.2×10⁹ FreeStyle 293F cells (Invitrogen Corp.) in the logarithmicgrowth phase were inoculated to a 3-L Fernbach Erlenmeyer Flask (CorningInc.), adjusted to 1.0×10⁶ cells/ml by dilution with FreeStyle 293expression medium (Invitrogen Corp.), and then shake-cultured at 90 rpmat 37° C. for 1 hour in an 8% CO₂ incubator. 3.6 mg of polyethyleneimine(Polysciences #24765) was dissolved in 20 ml of Opti-Pro SFM medium(Invitrogen Corp.). Next, each H chain expression vector (0.4 mg) andeach L chain expression vector (0.8 mg) prepared using PureLink HiPurePlasmid kit (Invitrogen Corp.) were suspended in 20 ml of Opti-Pro SFMmedium (Invitrogen Corp.). 20 ml of the expression vector/Opti-Pro SFMmixed solution was added to 20 ml of the polyethyleneimine/Opti-Pro SFMmixed solution, and the mixture was gently stirred, left for 5 minutes,and then added to the FreeStyle 293F cells. The cells wereshake-cultured at 90 rpm at 37° C. for 7 days in an 8% CO₂ incubator,and the obtained culture supernatant was filtered through DisposableCapsule Filter (ADVANTEC #CCS-045-E1H).

The human chimeric FR2-10 antibody obtained by the combination ofpCMA-G1/cFR2-10 and pCMA-LK/cFR2-10 was designated as “cFR2-10”. Thehuman chimeric FR2-13 antibody obtained by the combination ofpCMA-G1/cFR2-13 and pCMA-LK/cFR2-13 was designated as “cFR2-13”. Thehuman chimeric FR2-14 antibody obtained by the combination ofpCMA-G1/cFR2-14 and pCMA-LK/cFR2-14 was designated as “cFR2-14”.

4)-9-2 Purification of Human Chimeric Anti-FGFR2 Antibody

Each culture supernatant obtained in Example 4)-9-1 was purified by twosteps using rProtein A affinity chromatography (at 4 to 6° C.) andceramic hydroxyapatite (at room temperature). Buffer replacement stepsafter the rProtein A affinity chromatography purification and after theceramic hydroxyapatite purification were carried out at roomtemperature. First, 1100 to 1200 ml of the culture supernatant wasapplied to MabSelect SuRe (manufactured by GE Healthcare Bio-SciencesCorp., HiTrap column; volume 1 ml×2 connected) equilibrated with PBS.After entry of the whole culture solution into the column, the columnwas washed with 15 to 30 ml of PBS. Next, antibody-containing fractionswere collected by elution with a 2 M arginine hydrochloride solution (pH4.0). The fractions were buffer-replaced with 5 mM sodium phosphate, 50mM MES, and 20 mM NaCl (pH 6.5) using a desalting column (manufacturedby GE Healthcare Bio-Sciences Corp., HiTrap Desalting column; volume 5ml×2 connected). The buffer-replaced antibody solution was furtherapplied to a ceramic hydroxyapatite column (Bio-Rad Laboratories, Inc.,Bio-Scale CHT2-1 Hydroxyapatite Column; volume 2 ml) equilibrated with abuffer of 5 mM NaPi, 50 mM MES, and 20 mM NaCl (pH 6.5).Antibody-containing fractions were collected by linear concentrationgradient elution using sodium chloride. The fractions werebuffer-replaced with CBS (10 mM citrate buffer solution and 140 mMsodium chloride, pH 6.0) using a desalting column (manufactured by GEHealthcare Bio-Sciences Corp., HiTrap Desalting column; volume 5 ml×2connected). Finally, the fractions were concentrated and adjusted to anIgG concentration of 1.0 mg/ml or higher using Centrifugal UF FilterDevice VIVASPIN 20 (molecular weight cutoff: 30K, Sartorius Japan K.K.,at 4° C.), and used as a purified sample.

Example 5 In Vitro Activity of Human Chimeric Anti-FGFR2 Antibodies(cFR2-10, cFR2-13, and cFR2-14)

5)-1 Antigen Binding Activity of Human Chimeric Anti-FGFR2 Antibodies(cFR2-10, cFR2-13, and cFR2-14)

293α cells (described in Example 1)-6) were adjusted to 5×10⁵ cells/mlin a DMEM medium containing 10% FBS. pcDNA-DEST40-FGFR2 IIIb orpcDNA-DEST40-FGFR2 IIIc was transfected thereto using Lipofectamine 2000(manufactured by Invitrogen Corp.). The resulting cells were dispensedin an amount of 100 μl/well to a 96-well plate (manufactured by CorningInc.) and cultured overnight at 37° C. under 5% CO₂ conditions in a DMEMmedium containing 10% FBS. The obtained transfected cells were used inthe attached state in Cell-ELISA. After removal of the culturesupernatant, the cFR2-10, cFR2-13, or cFR2-14 antibody was added at afinal concentration of 2 μg/ml to the pcDNA-DEST40-FGFR2 IIIb- orpcDNA-DEST40-FGFR2 IIIc-transfected cells, and the plate was leftstanding at 4° C. for 1 hour. The cells in the wells were washed oncewith PBS containing 5% FBS. Then, Anti-Human IgG-Peroxidase antibodyproduced in goat (manufactured by Sigma-Aldrich Corp.) diluted 500-foldwith PBS containing 5% FBS was added thereto, and the plate was leftstanding at 4° C. for 1 hour. The cells in the wells were washed 5 timeswith PBS containing 5% FBS. Then, an OPD chromogenic solution (OPDsolution (o-phenylenediamine dihydrochloride (manufactured by Wako PureChemicals Industries, Ltd.) and H₂O₂ dissolved at concentrations of 0.4mg/ml and 0.6% (v/v), respectively, in 0.05 M trisodium citrate and 0.1M disodium hydrogen phosphate dodecahydrate, pH 4.5)) was added theretoat a concentration of 100 μl/well. Color reaction was performed withoccasional stirring and stopped by the addition of 1 M HCl at aconcentration of 100 μl/well. Then, the absorbance was measured at 490nm using a plate reader (ARVO; PerkinElmer, Inc.). As shown in FIG. 5,the cFR2-10 antibody selectively bound to FGFR2 IIIb, while the cFR2-13and cFR2-14 antibodies bound to both FGFR2 IIIb and FGFR2 IIIc.

5)-2 Signal-Neutralizing Effects of Human Chimeric Anti-FGFR2 Antibodies(cFR2-10, cFR2-13, and cFR2-14)

In order to evaluate the signal-neutralizing effects of the obtainedantibodies by the Elk1 luciferase reporter gene assay, 293α cells weretransfected with pcDNA-DEST40-FGFR2 IIIb or pcDNA-DEST40-FGFR2 IIIcconstructed in Example 1)-3-1 by the method shown in Example 1)-6-2, andcultured overnight at 37° C. under 5% CO₂ conditions. On the next day,the culture supernatant was removed, and the cells were thenpreincubated for 1 hour with the cFR2-10, cFR2-13, or cFR2-14 antibody(final concentration: 0.05 to 5 μg/ml) diluted with DMEM containing 2%FBS. Subsequently, a ligand human FGF7 (manufactured by R&D systems,Inc.) or human FGF9 (manufactured by PeproTech Inc.) was added at afinal concentration of 10 ng/ml to each well. After incubation for 6hours, cell lysates were prepared and assayed for firefly luciferaseactivity (specific signal) and Renilla luciferase activity (signal fornormalization) using Dual-luciferase reporter assay system (manufacturedby Promega Corp.). The firefly/Renilla ratio was calculated to normalizedata on each well. As shown in FIG. 6A, cFR2-10, cFR2-13, and cFR2-14inhibited ligand FGF7 dependent reporter activation in the FGFR2IIIb-expressing cells. As shown in FIG. 6B, cFR2-13 and cFR2-14inhibited ligand FGF9 dependent reporter activation in the FGFR2IIIc-expressing cells. These results demonstrated that these antibodieshave the effect of inhibiting the activation of FGFR2 by its ligand.

5)-3 ADCC Activity of Human Chimeric Anti-FGFR2 Antibodies (cFR2-10,cFR2-13, and cFR2-14)

5)-3-1 Preparation of Target Cell

293FT cells (Invitrogen Corp.) were cotransfected withpLenti6/V5-GW/lacZ and ViraPower™ Packaging Mix (Invitrogen Corp.)according to the attached protocols to prepare a recombinant lentivirusexpressing the β-galactosidase gene. 293T cells were infected by theobtained recombinant lentivirus according to the protocol of ViraPowerLentiviral Expression Systems (Invitrogen Corp.). Virus-infected cellswere selected using 10 μg/ml Blasticidin (Invitrogen Corp.) to obtain aline stably expressing β-galactosidase. These 293T cells stablyexpressing β-galactosidase were used as target cells in the assay ofADCC activity.

5)-3-2 Preparation of Target Cell

The stably β-galactosidase-expressing 293T cells (hereinafter, referredto as 293T-lacZ) obtained in Example 5)-3-1 were inoculated to a 225-cm²flask in an amount of 1×10⁷ cells/ml in a DMEM medium containing 10%FBS. After overnight culture at 37° C., pcDNA-DEST40-FGFR2 IIIb wastransfected to the cells using Lipofectamine 2000 (manufactured byInvitrogen Corp.), and the cells were cultured at 37° C. for 2 daysunder 5% CO₂ conditions in a DMEM medium containing 10% FBS and thendissociated and recovered from the flask using TrypLE Express(manufactured by Invitrogen Corp.). The cells were washed twice withphenol red-free RPMI1640 containing 5% FBS (hereinafter, referred to asa “medium for ADCC”). The number of live cells was counted by the trypanblue dye exclusion test. The cells were resuspended to 1×10⁵ cells/ml ina medium for ADCC and used as target cells.

5)-3-3 Preparation of Effector Cell

Uncharacterized Cryopreserved PBMC (manufactured by Cellular TechnologyLtd.) was suspended in a phenol red-free RPMI1640 medium (manufacturedby Invitrogen Corp.) containing 10% FBS, centrifuged, and thenresuspended. The number of live cells was counted by the trypan blue dyeexclusion test. After centrifugation, the medium was removed, and thecells were suspended and adjusted to a live cell density of 2.3×10⁶cells/ml in a medium for ADCC and used as effector cells.

5)-3-4 ADCC Assay

The 293T-lacZ cells prepared in Example 5)-3-2 were added at aconcentration of 50 μl/well to a 96-well U-bottomed microplate. ThecFR2-10, cFR2-13, cFR2-14, or human control antibody (hIgG) diluted to 1to 100 ng/ml (final concentration) with a medium for ADCC was addedthereto at a concentration of 50 μl/well, and the plate was leftstanding at 4° C. for 1 hour. The effector cells prepared in Example5)-3-3 were further added thereto at a concentration of 75 μl/well. Theplate was centrifuged at 1200 rpm at room temperature for 5 minutes,followed by overnight culture at 37° C. under 5% CO₂ conditions. On thenext day, 50 μl of the supernatant in each well was recovered into awhite plate (manufactured by Corning Inc.). A solution of β-Glo assaysystem (manufactured by Promega Corp.) was added thereto at aconcentration of 50 μl/well. The luminescence intensity was measuredusing a plate reader (ENVISION; manufactured by PerkinElmer, Inc.). Thepercentage of cells lysed by ADCC activity was calculated according tothe following expression:

Percentage of cells lysed(%)=(A−B)/(C−B)×100

A: Count of sample well

B: Average of spontaneous release (wells supplemented with neither theantibody nor the effector cells) counts (n=3). The same operation as inthe sample well was performed except that 50 μl and 75 μl of a mediumfor ADCC were added instead of the antibody and the effector cells,respectively.

C: Average of maximum release (wells containing target cells lysed in asurfactant) counts (n=3). 50 and 75 μl of a medium for ADCC were addedinstead of the antibody and the effector cells, respectively. For theassay, 175 μl of the β-Glo assay system solution was added to each wellcontaining the target cells and mixed therewith. A 100 μl aliquotthereof was added to a white plate to carry out the assay.

As shown in FIG. 7, cFR2-10, cFR2-13, and cFR2-14 had ADCC activityagainst the FGFR2 IIIb-expressing cells.

Example 6 In Vivo Antitumor Activity of Human Chimeric Anti-FGFR2Antibodies (cFR2-10, cFR2-13, and cFR2-14)

5×10⁶ cells of a human stomach cancer line SNU-16 (purchased from ATCC)were suspended in 50% Matrigel (purchased from Nippon Becton DickinsonCompany, Ltd.) and subcutaneously transplanted to the axillary region ofeach nude mouse (CAnN.Cg-Foxnl^(nu)/CrlCrlj, purchased from CharlesRiver Laboratories Japan Inc.). The mice were grouped according to theirtumor volumes. Seven, 11, 14, and 18 days after transplantation, eachhuman chimeric anti-FGFR2 antibody (cFR2-10, cFR2-13, or cFR2-14) wasintraperitoneally administered at a dose of 1.5 or 15 mg/kg to thecancer-bearing mice (n=8). The major axis and minor axis of thetransplanted tumor were measured twice a week using an electronicdigital caliper (manufactured by Mitsutoyo Corp.). The tumor volume wascalculated according to the following expression:

Tumor volume(mm³)=½×Minor axis(mm)×Minor axis(mm)×Major axis(mm)

The results on the cFR2-10 antibody are shown in FIG. 8-A. Thepercentage of tumor growth inhibition at 21 days after transplantation(final assay day) was 55% for the 1.5 mg/kg administration group and 35%for the 15 mg/kg administration group.

The results on the cFR2-13 antibody are shown in FIG. 8-B. Thepercentage of tumor growth inhibition at 21 days after transplantation(final assay day) was 57% for the 1.5 mg/kg administration group and 46%for the 15 mg/kg administration group.

The results on the cFR2-14 antibody are shown in FIG. 8-C. Thepercentage of tumor growth inhibition at 21 days after transplantation(final assay day) was 34% for the 1.5 mg/kg administration group and 53%for the 15 mg/kg administration group.

Example 7 Design of Humanized Version (hFR2-14) of Human ChimericAnti-FGFR2 Antibody (cFR2-14)

7)-1 Molecular Modeling of FR2-14 Variable Region

The molecular modeling of the cFR2-14 variable regions was carried outby a method generally known as homology modeling (Methods in Enzymology,203, 121-153, (1991)). The variable regions of FR2-14 determined abovewere compared with the primary sequences (three-dimensional structuresderived from X-ray crystal structures are available) of humanimmunoglobulin variable regions registered in Protein Data Bank (Nuc.Acid Res. 28, 235-242 (2000)). As a result, 1ZAN was selected because ithad the highest sequence homology to the light chain variable region ofcFR2-14. Also, 1CT8 was selected because of its highest sequencehomology to the heavy chain variable region of cFR2-14. Thethree-dimensional structures of framework regions were prepared as a“framework model” by combining the coordinates of 1ZAN and 1CT8corresponding to the light and heavy chains of cFR2-14. The CDRs ofcFR2-14 were assigned as clusters 11A, 7A, 10A, 10A, and 10A to CDRL1,CDRL2, CDRL3, CDRH1, and CDRH2, respectively, according to theclassification of Thornton et al. (J. Mol. Biol., 263, 800-815, (1996)).Its CDRH3 was classified into k(6)-according to the H3 rule (FEBSletter, 399, 1-8 (1996)). Subsequently, the typical conformation of eachCDR was incorporated into the framework model.

Finally, energy calculation for excluding disadvantageous interatomiccontact was conducted in order to obtain possible molecular models ofthe cFR2-14 variable regions in terms of energy. These procedures wereperformed using a commercially available protein three-dimensionalstructure prediction program Prime and conformation search programMacroModel (Schrodinger, LLC).

7)-2 Design of Amino Acid Sequence of Humanized FR2-14

The humanized FR2-14 antibody was constructed by a method generallyknown as CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033(1989)). An acceptor antibody was selected on the basis of the homologyof amino acids in framework regions.

The sequences of the cFR2-14 framework regions were compared with thesequences of all human frameworks registered in the Kabat database (Nuc.Acid Res., 29, 205-206 (2001)) of antibody amino acid sequences. As aresult, an FV/IL-2′CL antibody was selected as an acceptor due to its72% sequence homology as to framework regions. The amino acid residuesof the framework regions in FV/IL-2′CL were aligned with the amino acidresidues of the cFR2-14 framework regions to identify the positions ofamino acids that did not match there between. The positions of theseresidues were analyzed using the three-dimensional model of cFR2-14constructed above. Then, the donor residues to be grafted onto theacceptor were selected according to the criteria provided by Queen etal. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)).

Some donor residues thus selected were transferred to the acceptorantibody to construct the humanized FR2-14 sequence as described inExamples below.

In addition, 1 to 3 amino acid residues in each CDR of cFR2-14 weresubstituted by different amino acid residues to construct a humanizedFR2-14 sequence containing modified CDRs as described in Examples below.

7)-3 Humanization of FR2-14 Light Chain

7)-3-1 hFR2-14_L1 Type Light Chain:

A humanized FR2-14 light chain designed by the replacement of amino acidpositions 29 (alanine), 35 (leucine), 37 (glutamic acid), 38(threonine), 42 (glutamic acid), 62 (asparagine), 90 (glutamine), 92(serine), 94 (lysine), 96 (asparagine), 100 (serine), 103 (valine), 105(serine), 107 (phenylalanine), 121 (alanine), 125 (leucine), 127(leucine), and 130 (alanine) in the cFR2-14 light chain shown in SEQ IDNO: 48 of the Sequence Listing with serine, valine, aspartic acid,arginine, threonine, lysine, aspartic acid, threonine, threonine,serine, proline, phenylalanine, threonine, tyrosine, glutamine, valine,isoleucine, and threonine, respectively, was designated as an“hFR2-14_L1 type light chain”.

A nucleotide sequence encoding the hFR2-14_L1 type light chain is shownin SEQ ID NO: 72 of the Sequence Listing. Nucleotide positions 61 to 705encode a mature light chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_L1 type lightchain is shown in SEQ ID NO: 73 of the Sequence Listing. Amino acidpositions 21 to 235 represent a mature light chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 72 and 73are further described in FIGS. 80 and 81, respectively.

7)-4 Humanization of FR2-14 Heavy Chain

7)-4-1 hFR2-14_H1 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 62(proline), 63 (serine), 64 (threonine), 67 (isoleucine), 86 (lysine), 87(alanine), 94 (phenylalanine), 95 (serine), 101 (aspartic acid), 106(threonine), 110 (alanine) and 114 (phenylalanine) in the cFR2-14 heavychain shown in SEQ ID NO: 51 of the Sequence Listing with glutamine,valine, valine, lysine, valine, alanine, valine, arginine, alanine,glutamine, glycine, leucine, methionine, arginine, valine, serine,threonine, glutamic acid, arginine, threonine and tyrosine,respectively, was designated as an “hFR2-14_H1 type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H1 type heavy chain is shownin SEQ ID NO: 74 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H1 type heavychain is shown in SEQ ID NO: 75 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 74 and 75are further described in FIGS. 82 and 83, respectively.

7)-4-2 hFR2-14_H2 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 62(proline), 63 (serine), 64 (threonine), 67 (isoleucine), 86 (lysine), 87(alanine), 91 (valine), 94 (phenylalanine), 95 (serine), 101 (asparticacid), 106 (threonine), 110 (alanine) and 114 (phenylalanine) in thecFR2-14 heavy chain shown in SEQ ID NO: 51 of the Sequence Listing withglutamine, valine, valine, lysine, valine, alanine, valine, arginine,alanine, glutamine, glycine, leucine, methionine, arginine, valine,alanine, serine, threonine, glutamic acid, arginine, threonine andtyrosine, respectively, was designated as an “hFR2-14_H2 type heavychain”.

A nucleotide sequence encoding the hFR2-14_H2 type heavy chain is shownin SEQ ID NO: 76 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H2 type heavychain is shown in SEQ ID NO: 77 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 76 and 77are further described in FIGS. 84 and 85, respectively.

7)-4-3 hFR2-14_H3 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine) and 114(phenylalanine) in the cFR2-14 heavy chain shown in SEQ ID NO: 51 of theSequence Listing with glutamine, valine, valine, lysine, valine,alanine, valine, arginine, alanine, methionine, arginine, valine,serine, threonine, glutamic acid, arginine, threonine and tyrosine,respectively, was designated as an “hFR2-14_H3 type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H3 type heavy chain is shownin SEQ ID NO: 78 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H3 type heavychain is shown in SEQ ID NO: 79 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 78 and 79are further described in FIGS. 86 and 87, respectively.

7)-4-4 hFR2-14_H4 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 64(threonine), 67 (isoleucine), 86 (lysine), 87 (alanine),(phenylalanine), 95 (serine), 101 (aspartic acid), 106 (threonine), 110(alanine), 114 (phenylalanine) and 125 (threonine) in the cFR2-14 heavychain shown in SEQ ID NO: 51 of the Sequence Listing with glutamine,valine, valine, lysine, valine, alanine, valine, arginine, alanine,isoleucine, methionine, arginine, valine, serine, threonine, glutamicacid, arginine, threonine, tyrosine and alanine, respectively, wasdesignated as an “hFR2-14_H4 type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H4 type heavy chain is shownin SEQ ID NO: 80 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H4 type heavychain is shown in SEQ ID NO: 81 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 80 and 81are further described in FIGS. 88 and 89, respectively.

7)-4-5 hFR2-14_H5 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 118 (aspartic acid) in the cFR2-14 heavy chain shownin SEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and glutamic acid, respectively, was designated as an“hFR2-14_H5 type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H5 type heavy chain is shownin SEQ ID NO: 82 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H5 type heavychain is shown in SEQ ID NO: 83 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 82 and 83are further described in FIGS. 90 and 91, respectively.

7)-4-6 hFR2-14_H6 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 119 (glycine) in the cFR2-14 heavy chain shown inSEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and alanine, respectively, was designated as an “hFR2-14_H6type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H6 type heavy chain is shownin SEQ ID NO: 84 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H6 type heavychain is shown in SEQ ID NO: 85 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 84 and 85are further described in FIGS. 92 and 93, respectively.

7)-4-7 hFR2-14_H7 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 119 (glycine) in the cFR2-14 heavy chain shown inSEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and glutamic acid, respectively, was designated as an“hFR2-14_H7 type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H7 type heavy chain is shownin SEQ ID NO: 86 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H7 type heavychain is shown in SEQ ID NO: 87 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 86 and 87are further described in FIGS. 94 and 95, respectively.

7)-4-8 hFR2-14_H8 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 119 (glycine) in the cFR2-14 heavy chain shown inSEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and phenylalanine, respectively, was designated as an“hFR2-14_H8 type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H8 type heavy chain is shownin SEQ ID NO: 88 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H8 type heavychain is shown in SEQ ID NO: 89 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 88 and 89are further described in FIGS. 96 and 97, respectively.

7)-4-9 hFR2-14_H9 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 119 (glycine) in the cFR2-14 heavy chain shown inSEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and histidine, respectively, was designated as an “hFR2-14_H9type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H9 type heavy chain is shownin SEQ ID NO: 90 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H9 type heavychain is shown in SEQ ID NO: 91 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 90 and 91are further described in FIGS. 98 and 99, respectively.

7)-4-10 hFR2-14_H10 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 119 (glycine) in the cFR2-14 heavy chain shown inSEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and isoleucine, respectively, was designated as an “hFR2-14_H10type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H10 type heavy chain is shownin SEQ ID NO: 92 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H10 type heavychain is shown in SEQ ID NO: 93 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 92 and 93are further described in FIGS. 100 and 101, respectively.

7)-4-11 hFR2-14_H11 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 119 (glycine) in the cFR2-14 heavy chain shown inSEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and lysine, respectively, was designated as an “hFR2-14_H11type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H11 type heavy chain is shownin SEQ ID NO: 94 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H11 type heavychain is shown in SEQ ID NO: 95 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 94 and 95are further described in FIGS. 102 and 103, respectively.

7)-4-12 hFR2-14_H12 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 119 (glycine) in the cFR2-14 heavy chain shown inSEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and leucine, respectively, was designated as an “hFR2-14_H12type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H12 type heavy chain is shownin SEQ ID NO: 96 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H12 type heavychain is shown in SEQ ID NO: 97 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 96 and 97are further described in FIGS. 104 and 105, respectively.

7)-4-13 hFR2-14_H13 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 119 (glycine) in the cFR2-14 heavy chain shown inSEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and methionine, respectively, was designated as an “hFR2-14_H13type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H13 type heavy chain is shownin SEQ ID NO: 98 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H13 type heavychain is shown in SEQ ID NO: 99 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 98 and 99are further described in FIGS. 106 and 107, respectively.

7)-4-14 hFR2-14_H14 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 119 (glycine) in the cFR2-14 heavy chain shown inSEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and glutamine, respectively, was designated as an “hFR2-14_H14type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H14 type heavy chain is shownin SEQ ID NO: 100 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H14 type heavychain is shown in SEQ ID NO: 101 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 100 and101 are further described in FIGS. 108 and 109, respectively.

7)-4-15 hFR2-14_H15 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 119 (glycine) in the cFR2-14 heavy chain shown inSEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and arginine, respectively, was designated as an “hFR2-14_H15type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H15 type heavy chain is shownin SEQ ID NO: 102 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H15 type heavychain is shown in SEQ ID NO: 103 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 102 and103 are further described in FIGS. 110 and 111, respectively.

7)-4-16 hFR2-14_H16 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 119 (glycine) in the cFR2-14 heavy chain shown inSEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and valine, respectively, was designated as an “hFR2-14_H16type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H16 type heavy chain is shownin SEQ ID NO: 104 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H16 type heavychain is shown in SEQ ID NO: 105 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 104 and105 are further described in FIGS. 112 and 113, respectively.

7)-4-17 hFR2-14_H17 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 119 (glycine) in the cFR2-14 heavy chain shown inSEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and tryptophan, respectively, was designated as an “hFR2-14_H17type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H17 type heavy chain is shownin SEQ ID NO: 106 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H17 type heavychain is shown in SEQ ID NO: 107 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 106 and107 are further described in FIGS. 114 and 115, respectively.

7)-4-18 hFR2-14_H18 Type Heavy Chain:

A humanized FR2-14 heavy chain designed by the replacement of amino acidpositions 22 (lysine), 24 (leucine), 30 (leucine), 31 (valine), 39(leucine), 43 (threonine), 56 (leucine), 57 (lysine), 59 (valine), 67(isoleucine), 86 (lysine), 87 (alanine), 94 (phenylalanine), 95(serine), 101 (aspartic acid), 106 (threonine), 110 (alanine), 114(phenylalanine) and 119 (glycine) in the cFR2-14 heavy chain shown inSEQ ID NO: 51 of the Sequence Listing with glutamine, valine, valine,lysine, valine, alanine, valine, arginine, alanine, methionine,arginine, valine, serine, threonine, glutamic acid, arginine, threonine,tyrosine and tyrosine, respectively, was designated as an “hFR2-14_H18type heavy chain”.

A nucleotide sequence encoding the hFR2-14_H18 type heavy chain is shownin SEQ ID NO: 108 of the Sequence Listing. Nucleotide positions 58 to1401 encode a mature heavy chain produced by the cleavage of the signalsequence. Also, the amino acid sequence of the hFR2-14_H18 type heavychain is shown in SEQ ID NO: 109 of the Sequence Listing. Amino acidpositions 20 to 467 represent a mature heavy chain produced by thecleavage of the signal sequence. The sequences of SEQ ID NOs: 108 and109 are further described in FIGS. 116 and 117, respectively.

Example 8 Obtainment and Expression of Humanized Antibody (hFR2-14) ofRat Anti-Human FGFR2 Antibody FR2-14

8)-1 Construction of Light Chain Expression Vector for HumanizedAntibody (hFR2-14) of Rat Anti-Human FGFR2 Antibody FR2-14

8)-1-1 Construction of hFR2-14_L1 Type Light Chain Expression Vector

A DNA comprising a gene encoding the hFR2-14_L1 type light chainvariable region shown in amino acid positions 21 to 130 of SEQ ID NO: 73was synthesized and cleaved with a restriction enzyme BsiWI. Theresulting DNA fragment was inserted to the restriction enzymeBsiWI-cleaved site of the general-purpose vector (pCMA-LK) for chimericand humanized antibody light chain expression to construct an hFR2-14_L1type light chain expression vector. The obtained expression vector wasdesignated as “pCMA-LK/hFR2-14_L1”.

8)-2 Construction of Heavy Chain Expression Vector for HumanizedAntibody (hFR2-14) of Rat Anti-Human FGFR2 Antibody FR2-14

8)-2-1 Construction of hFR2-14_H1, hFR2-14_H3, and hFR2-14_H4 Type HeavyChain Expression Vectors

A DNA comprising a gene encoding each of the hFR2-14_H1, hFR2-14_H3, andhFR2-14_H4 type heavy chain variable regions shown in amino acidpositions 20 to 137 of SEQ ID NO: 75, amino acid positions 20 to 137 ofSEQ ID NO: 79, and amino acid positions 20 to 137 of SEQ ID NO: 81,respectively, of the Sequence Listing was synthesized and cleaved with arestriction enzyme BlpI. The resulting DNA fragment was inserted to therestriction enzyme BlpI-cleaved site of the general-purpose vector(pCMA-G1) for humanized antibody heavy chain expression to constructhFR2-14_H1, hFR2-14_H3, and hFR2-14_H4 type heavy chain expressionvectors. The obtained expression vectors were designated as“pCMA-G1/hFR2-14_H1”, “pCMA-G1/hFR2-14_H3”, and “pCMA-G1/hFR2-14_H4”,respectively.

8)-2-2 Construction of hFR2-14_H2 Type Heavy Chain Expression Vector

An hFR2-14_H2 type heavy chain expression vector was constructed usingpCMA-G1/hFR2-14_H1 constructed in Example 8)-2-1 as a template, a primerset shown below, and QuikChange XL Site-Directed Mutagenesis Kit(Agilent Technologies, Inc.). The obtained expression vector wasdesignated a “pCMA-G1/hFR2-14_H2”. The nucleotide sequence of thehFR2-14_H2 type heavy chain is shown in SEQ ID NO: 76 of the SequenceListing, and its amino acid sequence is shown in SEQ ID NO: 77.

Primer Set

(VH3A-F: SEQ ID NO: 110 of the Sequence Listing)5′-ggcagagtgaccctgaccgccgacaagagcaccagcacc-3′(VH3A-R: SEQ ID NO: 111 of the Sequence Listing)5′-ggtgctggtgctcttgtcggcggtcagggtcactctgcc-3′

8)-2-3 Construction of hFR2-14_H5 Type Heavy Chain Expression Vector

The hFR2-14_H3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_H5 type heavy chain expression vector. Theobtained expression vector was designated as “pCMA-G1/hFR2-14_H5”.

Primer Set:

5′-GAGGGCTACGGCGACTGGTTCACATAC-3′ (H5-F) 5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R, which is  used as a common primer in the description below)

8)-2-4 Construction of hFR2-14_H6 Type Heavy Chain Expression Vector

The hFR2-14_H3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_H6 type heavy chain expression vector. Theobtained expression vector was designated as “pCMA-G1/hFR2-14_H6”.

Primer Set:

5′-GACGCCTACGGCGACTGGTTCACATAC-3′ (H6-F) 5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R)

8)-2-5 Construction of hFR2-14_H7 Type Heavy Chain Expression Vector

The hFR2-14_H3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_H7 type heavy chain expression vector. Theobtained expression vector was designated as “pCMA-G1/hFR2-14_H7”.

Primer Set:

5′-GACGAGTACGGCGACTGGTTCACATAC-3′ (H7-F)  5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R)

8)-2-6 Construction of hFR2-14_H8 Type Heavy Chain Expression Vector

The hFR2-14_H3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_H8 type heavy chain expression vector. Theobtained expression vector was designated as “pCMA-G1/hFR2-14_H8”.

Primer Set:

5′-GACTTCTACGGCGACTGGTTCACATAC-3′ (H8-F) 5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R)

8)-2-7 Construction of hFR2-14_H9 Type Heavy Chain Expression Vector

The hFR2-14_H3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_H9 type heavy chain expression vector. Theobtained expression vector was designated as “pCMA-G1/hFR2-14_H9”.

Primer Set:

5′-GACCACTACGGCGACTGGTTCACATAC-3′ (H9-F) 5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R)

8)-2-8 Construction of hFR2-14_H10 Type Heavy Chain Expression Vector

The hFR2-14_H3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_H10 type heavy chain expression vector.The obtained expression vector was designated as “pCMA-G1/hFR2-14_H10”.

Primer Set:

5′-GACATCTACGGCGACTGGTTCACATAC-3′ (H10-F) 5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R)

8)-2-9 Construction of hFR2-14_H11 Type Heavy Chain Expression Vector

The hFR2-14_H3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_H11 type heavy chain expression vector.The obtained expression vector was designated as “pCMA-G1/hFR2-14_H11”.

Primer Set:

5′-GACAAGTACGGCGACTGGTTCACATAC-3′ (H11-F) 5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R)

8)-2-10 Construction of hFR2-14_H12 Type Heavy Chain Expression Vector

The hFR2-14_H3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_hH12 type heavy chain expression vector.The obtained expression vector was designated as “pCMA-G1/hFR2-14_H12”.

Primer Set:

5′-GACCTGTACGGCGACTGGTTCACATAC-3′ (H12-F) 5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R)

8)-2-11 Construction of hFR2-14_hH13 Type Heavy Chain Expression Vector

The hFR2-14_hH3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_hH13 type heavy chain expression vector.The obtained expression vector was designated as “pCMA-G1/hFR2-14_H13”.

Primer Set:

5′-GACATGTACGGCGACTGGTTCACATAC-3′ (H13-F) 5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R)

8)-2-12 Construction of hFR2-14_hH14 Type Heavy Chain Expression Vector

The hFR2-14_hH3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_hH14 type heavy chain expression vector.The obtained expression vector was designated as “pCMA-G1/hFR2-14_H14”.

Primer Set:

5′-GACCAGTACGGCGACTGGTTCACATAC-3′ (H14-F) 5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R)

8)-2-13 Construction of hFR2-14_hH15 Type Heavy Chain Expression Vector

The hFR2-14_hH3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_hH15 type heavy chain expression vector.The obtained expression vector was designated as “pCMA-G1/hFR2-14_H15”.

Primer Set:

5′-GACCGGTACGGCGACTGGTTCACATAC-3′ (H15-F) 5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R)

8)-2-14 Construction of hFR2-14_hH16 Type Heavy Chain Expression Vector

The hFR2-14_hH3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_hH16 type heavy chain expression vector.The obtained expression vector was designated as “pCMA-G1/hFR2-14_H16”.

Primer Set:

5′-GACGTGTACGGCGACTGGTTCACATAC-3′ (H16-F) 5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R)

8)-2-15 Construction of hFR2-14_hH17 Type Heavy Chain Expression Vector

The hFR2-14_hH3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_hH17 type heavy chain expression vector.The obtained expression vector was designated as “pCMA-G1/hFR2-14_H17”.

Primer Set:

5′-GACTGGTACGGCGACTGGTTCACATAC-3′ (H17-F) 5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R)

8)-2-16 Construction of hFR2-14_hH18 Type Heavy Chain Expression Vector

The hFR2-14_hH3 type heavy chain expression vector pCMA-G1/hFR2-14_H3prepared in Example 8)-2-1 was used as a template and mutated using aprimer set described below and KOD-Plus-Mutagenesis Kit (Toyobo Co.,Ltd.) to construct an hFR2-14_hH18 type heavy chain expression vector.The obtained expression vector was designated as “pCMA-G1/hFR2-14_H18”.

Primer Set:

5′-GACTACTACGGCGACTGGTTCACATAC-3′ (H18-F) 5′-GGTGGCGCAGTAGTACACGGCGGT-3′(H-R)

8)-3 Preparation of Humanized FR2-14 Antibody (FreeStyle 293F Cell)

8)-3-1 Production of Humanized FR2-14 Antibody

FreeStyle 293F cells (Invitrogen Corp.) were subcultured and culturedaccording to the manual.

1.2×10⁹ FreeStyle 293F cells (Invitrogen Corp.) in the logarithmicgrowth phase were inoculated to a 3-L Fernbach Erlenmeyer Flask (CorningInc.), adjusted to 1.0×10⁶ cells/mL by dilution with FreeStyle 293expression medium (Invitrogen Corp.), and then shake-cultured at 90 rpmat 37° C. for 1 hour in an 8% CO₂ incubator. 3.6 mg of polyethyleneimine(Polysciences #24765) was dissolved in 20 ml of Opti-Pro SFM medium(Invitrogen Corp.). Next, each H chain expression vector (0.4 mg) andthe L chain expression vector (0.8 mg) prepared using PureLink HiPurePlasmid kit (Invitrogen Corp.) were suspended in 20 ml of Opti-Pro SFMmedium (Invitrogen Corp.). 20 ml of the expression vector/Opti-Pro SFMmixed solution was added to 20 ml of the polyethyleneimine/Opti-Pro SFMmixed solution, and the mixture was gently stirred, further left for 5minutes, and then added to the FreeStyle 293F cells. The cells wereshake-cultured at 90 rpm at 37° C. for 7 days in an 8% CO₂ incubator,and the obtained culture supernatant was filtered through DisposableCapsule Filter (ADVANTEC #CCS-045-E1H).

The humanized FR2-14 antibodies obtained by the combination ofpCMA-G1/hFR2-14_H5, pCMA-G1/hFR2-14_H6, pCMA-G1/hFR2-14_H7,pCMA-G1/hFR2-14_H8, pCMA-G1/hFR2-14_H9, pCMA-G1/hFR2-14_H10,pCMA-G1/hFR2-14_H11, pCMA-G1/hFR2-14_H12, pCMA-G1/hFR2-14_H13,pCMA-G1/hFR2-14_H14, pCMA-G1/hFR2-14_H15, pCMA-G1/hFR2-14_H16,pCMA-G1/hFR2-14_H17 and pCMA-G1/hFR2-14_H18 with pCMA-LK/hFR2-14_L1 weredesignated as “hFR2-14_H5/L1”, “hFR2-14_H6/L1”, “hFR2-14_H7/L1”,“hFR2-14_H8/L1”, “hFR2-14_H9/L1”, “hFR2-14_H10/L1”, “hFR2-14_H11/L1”,“hFR2-14_H12/L1”, “hFR2-14_H13/L1”, “hFR2-14_H14/L1”, “hFR2-14_H15/L1”,“hFR2-14_H16/L1”, “hFR2-14_H17/L1” and “hFR2-14_H18/L1, respectively.

8)-3-2 Purification of Humanized FR2-14 Antibody

Each antibody was purified from the culture supernatant obtained inExample 8)-3-1 by two steps using rProtein A affinity chromatography (at4 to 6° C.) and ceramic hydroxyapatite (at room temperature). Bufferreplacement steps after the rProtein A affinity chromatographypurification and after the ceramic hydroxyapatite purification werecarried out at 4 to 6° C. First, the culture supernatant was applied toMabSelect SuRe (manufactured by GE Healthcare Bio-Sciences Corp., HiTrapcolumn) equilibrated with PBS. After entry of the whole culture solutionin the column, the column was washed with PBS in an amount at leasttwice the column volume. Next, antibody-containing fractions werecollected by elution with a 2 M arginine hydrochloride solution (pH4.0). The fractions were buffer-replaced with PBS by dialysis (ThermoFisher Scientific Inc., Slide-A-Lyzer Dialysis Cassette) and thendiluted 5-fold with a buffer of 5 mM sodium phosphate and 50 mM MES (pH7.0). The resulting antibody solution was applied to a ceramichydroxyapatite column (Bio-Rad Laboratories, Inc., Bio-Scale CHT Type-IHydroxyapatite Column) equilibrated with a buffer of 5 mM NaPi, 50 mMMES, and 30 mM NaCl (pH 7.0). Antibody-containing fractions werecollected by linear concentration gradient elution using sodiumchloride. The fractions were buffer-replaced with HBSor (25 mM histidineand 5% sorbitol, pH 6.0) by dialysis (Thermo Fisher Scientific Inc.,Slide-A-Lyzer Dialysis Cassette). Finally, the fractions wereconcentrated and adjusted to an IgG concentration of 25 mg/ml or higherusing Centrifugal UF Filter Device VIVASPIN 20 (molecular weight cutoff:UF10K, Sartorius Japan K.K., at 4° C.), and used as a purified sample.

Example 9 Preparation of Humanized FR2-14 Antibody with Regulated SugarChain Modification

The humanized antibody comprising the heavy chain comprising amino acidpositions 20 to 467 of the amino acid sequence represented by SEQ ID NO:97 (FIG. 105), and the light chain comprising amino acid positions 21 to235 of the amino acid sequence represented by SEQ ID NO: 73 (FIG. 81)was defucosylated according to a method known in the art to regulate thesugar chain modification of the antibody protein. The obtained antibodywas designated as hFR2-14_H19/L1. This modified form was subjected tomass spectrometry. As a result, the peak of a fucose-containing H chainwas equal to or lower than the detection limit. In the presentinvention, the antibody with regulated sugar chain modification, such ashFR2-14_H19/L1, is also referred to as an “antibody” or a “modified formof the antibody”.

Example 10 Physical Property Evaluation of Humanized Anti-Human FGFR2Antibody (hFR2-14)

10)-1 Biacore Assay of Antigen Binding Activity of Humanized Anti-HumanFGFR2 Antibody (hFR2-14)

The antibody was assayed for its dissociation constant for an antigen(rhFGFR2 alpha (IIIb) Fc chimera or rhFGFR2 alpha (IIIc) Fc chimera)using Biacore 3000 (GE Healthcare Bio-Sciences Corp.) by the capturemethod, which involves capturing the antibody as a ligand onto animmobilized anti-human IgG(Fab) antibody and assaying the antigen as ananalyte. Approximately 5000 RU of the anti-human IgG(Fab) antibody(Human Fab capture kit, GE Healthcare Bio-Sciences Corp.) was covalentlybound to a sensor chip CM5 (BIAcore, Inc.) by the amine coupling method.Similarly, this antibody was immobilized onto a reference cell. Therunning buffer used was HBS-EP+ (10 mM HEPES (pH 7.4), 0.15 M NaCl, 3 mMEDTA, and 0.05% Surfactant P20). A 1 μg/mL antibody solution of thepurified antibody was added onto the anti-human IgG(Fab)antibody-immobilized chip, at a flow rate of 10 μL/min for 60 seconds orthe culture supernatant containing the antibody was added for 60seconds. Then, serial dilutions (0.3 to 500 nM) of the antigen wereadded thereto at a flow rate of 30 μl/min for 180 seconds. Subsequently,the dissociation phase was monitored for 300 seconds. 10 mM Gly-HCl (pH2.1) was added twice thereto as a regenerating solution at a flow rateof 10 μl/min for 60 seconds. The data was analyzed using the Bivalentbinding model of analytical software (BIAevaluation software, version4.1) to calculate an association rate constant kon, a dissociation rateconstant koff, and a dissociation constant (KD; KD=koff/kon).

10)-1-1 Binding Activity Evaluation of 4 Types of Humanized Anti-FGFR2Antibodies (hFR2-14_H1/L1 to hFR2-14_H4/L1) and Human ChimericAnti-FGFR2 Antibody (cFR2-14)

Four types of humanized anti-FGFR2 antibodies (hFR2-14_H1/L1 tohFR2-14_H4/L1) and the human chimeric anti-FGFR2 antibody (cFR2-14) wereexpressed and purified by the methods of Examples 8)-3 and 4)-9 andevaluated for their binding activity against each human FGFR2 variantprotein by the method shown in Example 10)-1. The Biacore assay resultsare shown in FIG. 122.

10)-1-2 Binding Activity Evaluation of 15 Types of Humanized Anti-FGFR2Antibodies (hFR2-14_H3/L1 and hFR2-14_H5/L1 to hFR2-14_H18/L1)

Fifteen types of humanized anti-FGFR2 antibodies (hFR2-14_H3/L1 andhFR2-14_H5/L1 to hFR2-14_H18/L1) were expressed by the method of Example8)-3, and the culture supernatant containing each antibody was used inthe evaluation of binding activity against the FGFR2 IIIc protein by themethod shown in Example 10)-1. The Biacore assay results are shown inFIG. 123.

10)-2 Study on Selective Binding Activity of Humanized Anti-Human FGFR2Antibodies (hFR2-14_H3/L1, hFR2-14_H8/L1, and hFR2-14_H12/L1) AgainstHuman FGFR2

10)-2-1 Construction of Human FGFR1 IIIb Expression Vector(pcDNA-DEST40-FGFR1 IIIb)

A cDNA encoding a human FGFR1 IIIb variant protein (protein comprisingthe amino acid sequence of the FGFR1 IIIb domain (AAB19502) between anamino acid sequence of positions 1 to 310 and an amino acid sequence ofpositions 359 to 820 of isoform 2 (NP_(—)056934)) was cloned into apcDNA-DEST40 vector to construct pcDNA-DEST40-FGFR1 IIIb.

10)-2-2 Cell-ELISA

Various human FGFR expression vectors constructed in Examples 1)-3-1,2)-1-1, and 10)-2-1 were separately transfected to 293α cells (describedin Example 1)-6) using Lipofectamine 2000 (manufactured by LifeTechnologies Corp.). The resulting cells were dispensed in an amount of100 μl/well to a 96-well plate (manufactured by Corning Inc.) andcultured overnight at 37° C. under 5% CO₂ conditions in a DMEM mediumcontaining 10% FBS. After removal of the culture supernatant, a solutionof hFR2-14_H3/L1, hFR2-14_H8/L1, or hFR2-14_H12/L1 diluted to 3 μg/mlwith PBS containing 5% FBS was added at a concentration of 50 μl/well tothe plate, and the plate was left standing at 4° C. for 1 hour. Thecells in the wells were washed twice with PBS containing 5% FBS. Then,Anti-human IgG-Peroxidase conjugate antibody produced in goat(manufactured by Sigma-Aldrich Corp.) and diluted 2000-fold with PBScontaining 5% FBS was added thereto, and the plate was left standing at4° C. for 1 hour. The cells in the wells were washed 3 times with PBScontaining 5% FBS. Then, an OPD chromogenic solution (OPD solution(o-phenylenediamine dihydrochloride (manufactured by Wako Pure ChemicalsIndustries, Ltd.) and H₂O₂ dissolved at concentrations of 0.4 mg/ml and0.6% (v/v), respectively, in 0.05 M trisodium citrate and 0.1 M disodiumhydrogen phosphate dodecahydrate, pH 4.5)) was added thereto at aconcentration of 100 μl/well. Color reaction was performed withoccasional stirring and stopped by the addition of 1 M HCl at aconcentration of 100 μl/well. Then, the absorbance was measured at 490nm using a plate reader (ARVO; PerkinElmer, Inc.). As seen from FIG.124, the hFR2-14_H3/L1, hFR2-14_H8/L1, and hFR2-14_H12/L1 antibodieswere shown to specifically bind to both human FGFR2 IIIb and FGFR2 IIIc.

10)-3 Thermal Stability Assay of Humanized Anti-Human FGFR2 Antibody(hFR2-14) Using Differential Scanning Calorimetry (DSC)

The thermal stability was assayed using differential scanningcalorimetry (DSC). Each sample was dissolved at a concentration of 0.5mg/mL in an HBSor buffer solution (prepared to contain 25 mM histidine(pH 6.0) and 5% sorbitol), and 400 μL of the sample solution was used inthe DSC assay. The DSC assay conditions were set as follows: an initialtemperature of 20° C.; a final temperature of 100° C.; a rate oftemperature rise of 200° C./hour; a filtering time of 2 seconds; and afeedback mode of Low. The reference solution used was HBSor.VP-Capillary DSC Platform manufactured by GE Healthcare Bio-SciencesCorp. (USA) was used as a DSC assay apparatus in all experiments.Baseline correction was conducted by the subtraction of the baseline(scan curve obtained from the reference solution also charged into asample cell) from a scan curve obtained from the sample solution. Next,concentration calibration was conducted using molar concentrationcalculated from the molecular weight of each sample. FIGS. 125A and 125Bshow the thermograms of various humanized FGFR2 antibodies. The thermaldenaturation midpoint Tm is defined as the temperature at which themaximum peak in each thermogram exhibited a peak top. As shown in FIG.125C, the hFR2-14_H1/L1 antibody had a Tm value of 87.6° C. ThehFR2-14_H2/L1 antibody had a Tm value of 87.2° C. The hFR2-14_H3/L1antibody had a Tm value of 79.5° C. The hFR2-14_H4/L1 antibody had a Tmvalue of 81.6° C. The hFR2-14_H5/L1 antibody had a Tm value of 77.2° C.The hFR2-14_H8/L1 antibody had a Tm value of 81.0° C. The hFR2-14_H9/L1antibody had a Tm value of 78.8° C. The hFR2-14_H11/L1 antibody had a Tmvalue of 80.3° C. The hFR2-14_H12/L1 antibody had a Tm value of 82.2° C.The hFR2-14_H19/L1 antibody had a Tm value of 82.2° C.

10)-4 Binding Stability Test of Humanized Anti-Human FGFR2 Antibody(hFR2-14) Using Biacore

The humanized anti-human FGFR2 antibody (hFR2-14) was evaluated for itsantigen binding stability by a method described below.

Various humanized anti-FGFR2 antibodies (hFR2-14_H1/L1 to hFR2-14_H5/L1,hFR2-14_H8/L1, hFR2-14_H9/L1, hFR2-14_H11/L1, hFR2-14_H12/L1, andhFR2-14_H19/L1) and the human chimeric anti-FGFR2 antibody (cFR2-14)were expressed and purified by the methods of Examples 8)-3, 9), and4)-9 and each dissolved at a concentration of 20 mg/mL in an HBSorbuffer solution (prepared to contain 25 mM histidine (pH 6.0) and 5%sorbitol). The solutions were heated to 40° C. for 4 weeks to preparedegraded analytes. The analytes were assayed for their binding activitybefore and after degradation by the method shown in Example 10)-1 usingBiacore. The Biacore assay results are shown in FIG. 126.

Example 11 Signal-Neutralizing Effect of Humanized Anti-Human FGFR2Antibody (hFR2-14)

11)-1 Signal-Neutralizing Effects of Humanized Anti-FGFR2 Antibodies(hFR2-14_H1/L1, hFR2-14_H2/L1, hFR2-14_H3/L1, and hFR2-14_H4/L1) andHuman Chimeric Anti-FGFR2 Antibody (cFR2-14)

In order to evaluate the signal-neutralizing effects of the humanizedantibodies by the Elk1 luciferase reporter gene assay, 293α cells werecotransfected with pcDNA-DEST40-FGFR2 IIIb or pcDNA-DEST40-FGFR2 IIIc,pFA2-Elk1 (manufactured by Stratagene Corp.), pFR-Luc2CP, andpGL4.74[hRluc/TK] (manufactured by Promega Corp.) by the method shown inExample 1)-6-2, and cultured overnight at 37° C. under 5% CO₂conditions. On the next day, the culture supernatant was removed, andthe cells were then preincubated for 1 hour with the hFR2-14_H1/L1,hFR2-14_H2/L1, hFR2-14_H3/L1, hFR2-14_H4/L1, or cFR2-14 antibody dilutedwith DMEM containing 2% FBS. Subsequently, a ligand (human FGF7 or humanFGF9, manufactured by PeproTech Inc.) was added at a final concentrationof 10 ng/ml to each well. After incubation for 6 hours, fireflyluciferase activity (specific signal) and Renilla luciferase activity(signal for normalization) were assayed using Dual-Glo Luciferase AssaySystem (manufactured by Promega Corp.). The firefly/Renilla ratio wascalculated to normalize data on each well. As shown in FIG. 127A,hFR2-14_H1/L1, hFR2-14_H2/L1, hFR2-14_H3/L1, hFR2-14_H4/L1, and cFR2-14inhibited ligand FGF7 dependent reporter activation in the FGFR2IIIb-expressing cells. As shown in FIG. 127B, hFR2-14_H1/L1,hFR2-14_H2/L1, hFR2-14_H3/L1, hFR2-14_H4/L1, and cFR2-14 inhibitedligand FGF9 dependent reporter activity in the FGFR2 IIIc-expressingcells. These results demonstrated that these antibodies have the effectof inhibiting the activation of FGFR2 by its ligand.

11)-2 Signal-Neutralizing Effects of Humanized Anti-FGFR2 Antibodies(hFR2-14_H3/L1, hFR2-14_H5/L1, hFR2-14_H6/L1, hFR2-14_H7/L1,hFR2-14_H8/L1, hFR2-14_H9/L1, hFR2-14_H10/L1, hFR2-14_H11/L1,hFR2-14_H12/L1, hFR2-14_H13/L1, hFR2-14_H14/L1, hFR2-14_H15/L1,hFR2-14_H16/L1, hFR2-14_H17/L1, and hFR2-14_H18/L1)

In order to evaluate the signal-neutralizing effects of the humanizedantibodies by the Elk1 luciferase reporter gene assay, 293α cells werecotransfected with pcDNA-DEST40-FGFR2 IIIb or pcDNA-DEST40-FGFR2 IIIc,pFA2-Elk1 (manufactured by Stratagene Corp.), pFR-Luc2CP, andpGL4.74[hRluc/TK] (manufactured by Promega Corp.) by the method shown inExample 1)-6-2, and cultured overnight at 37° C. under 5% CO₂conditions. On the next day, the culture supernatant was removed, andthe cells were then preincubated for 1 hour with the culture supernatantof the 293 FreeStyle cells (manufactured by Invitrogen Corp.) producingthe antibodies hFR2-14_H3/L1, hFR2-14_H5/L1, hFR2-14_H6/L1,hFR2-14_H7/L1, hFR2-14_H8/L1, hFR2-14_H9/L1, hFR2-14_H10/L1,hFR2-14_H11/L1, hFR2-14_H12/L1, hFR2-14_H13/L1, hFR2-14_H14/L1,hFR2-14_H15/L1, hFR2-14_H16/L1, hFR2-14_H17/L1, and hFR2-14_H18/L1(prepared in Example 8)-3-1) diluted with DMEM containing 2% FBS.Subsequently, a ligand (human FGF7, manufactured by R&D systems, Inc.)was added at a final concentration of 10 ng/ml to each well. Afterincubation for 6 hours, firefly luciferase activity (specific signal)and Renilla luciferase activity (signal for normalization) were assayedusing Dual-Glo Luciferase Assay System (manufactured by Promega Corp.).The firefly/Renilla ratio was calculated to normalize data on each well.As shown in FIGS. 128A, 128B, and 128C, hFR2-14_H3/L1, hFR2-14_H5/L1,hFR2-14_H6/L1, hFR2-14_H7/L1, hFR2-14_H8/L1, hFR2-14_H9/L1,hFR2-14_H10/L1, hFR2-14_H11/L1, hFR2-14_H12/L1, hFR2-14_H13/L1,hFR2-14_H14/L1, hFR2-14_H15/L1, hFR2-14_H16/L1, hFR2-14_H17/L1, andhFR2-14_H18/L1 inhibited ligand FGF7 dependent reporter activation inthe FGFR2 IIIb-expressing cells.

11)-3 Signal-Neutralizing Effects of Humanized Anti-FGFR2 Antibodies(hFR2-14_H12/L1 and hFR2-14_H19/L1)

In order to evaluate the signal-neutralizing effects of the humanizedantibodies by the Elk1 luciferase reporter gene assay, 293α cells werecotransfected with pcDNA-DEST40-FGFR2 IIIb or pcDNA-DEST40-FGFR2 IIIc,pFA2-Elk1 (manufactured by Stratagene Corp.), pFR-Luc2CP, andpGL4.74[hRluc/TK] (manufactured by Promega Corp.) by the method shown inExample 1)-6-2, and cultured overnight at 37° C. under 5% CO₂conditions. On the next day, the culture supernatant was removed, andthe cells were then preincubated for 1 hour with the hFR2-14_H12/L1 orhFR2-14_H19/L1 antibody (prepared in Examples 8) and 9)) diluted withDMEM containing 2% FBS. Subsequently, a ligand human FGF7 (manufacturedby R&D systems, Inc.) or human FGF9 (manufactured by PeproTech Inc.) wasadded at a final concentration of 10 ng/ml to each well. Afterincubation for 6 hours, firefly luciferase activity (specific signal)and Renilla luciferase activity (signal for normalization) were assayedusing Dual-Glo Luciferase Assay System (manufactured by Promega Corp.).The firefly/Renilla ratio was calculated to normalize data on each well.As shown in FIG. 129A, hFR2-14_H12/L1 and hFR2-14_H19/L1 inhibitedligand FGF7 dependent reporter activation in the FGFR2 IIIb-expressingcells. As shown in FIG. 129B, hFR2-14_H12/L1 and hFR2-14_H19/L1inhibited ligand FGF9 dependent reporter activity in the FGFR2IIIc-expressing cells. These results demonstrated that these antibodieshave the effect of inhibiting the activation of FGFR2 by its ligand.

Example 12 ADCC Activity of Humanized Anti-Human FGFR2 Antibody(hFR2-14)

12)-1 ADCC Activity of Humanized Anti-FGFR2 Antibodies (hFR2-14_H1/L1,hFR2-14_H2/L1, hFR2-14_H3/L1, and hFR2-14_H4/L1) and Human ChimericAnti-FGFR2 Antibody (cFR2-14) Against FGFR2-Overexpressing Cell

The FGFR2 IIIb-expressing 293T-lacZ cells prepared by the method ofExample 5)-3-2 were added at a concentration of 50 μl/well to a 96-wellU-bottomed microplate. hFR2-14_H1/L1, hFR2-14_H2/L1, hFR2-14_H3/L1,hFR2-14_H4/L1, cFR2-14, or human IgG diluted to 1 to 100 ng/ml (finalconcentration) with a medium for ADCC described in Example 5)-3-2 wasadded thereto at a concentration of 50 μl/well, and the plate was leftstanding at 4° C. for 1 hour. The effector cells of Example 5)-3-3 werefurther added thereto at a concentration of 75 μl/well. The plate wascentrifuged at 1200 rpm at room temperature for 5 minutes, followed byovernight culture at 37° C. under 5% CO₂ conditions. On the next day, 50μl of the supernatant in each well was recovered into a white plate(manufactured by Corning Inc.). A solution of β-Glo assay system(manufactured by Promega Corp.) was added thereto at a concentration of50 μl/well. The luminescence intensity was measured using a plate reader(ENVISION; manufactured by PerkinElmer, Inc.). The percentage of cellslysed by ADCC activity was calculated according to the followingexpression:

Percentage of cells lysed(%)=(A−B)/(C−B)×100

A: Count of sample well

B: Average of spontaneous release (wells supplemented with neither theantibody nor the effector cells) counts (n=3). The same operation as inthe sample well was performed except that 50 μl and 75 μl of a mediumfor ADCC were added instead of the antibody and the effector cells,respectively.

C: Average of maximum release (wells containing target cells lysed in asurfactant) counts (n=3). 50 μl and 75 μl of a medium for ADCC wereadded instead of the antibody and the effector cells, respectively. Forthe assay, 175 μl of the β-Glo assay system solution was added to eachwell containing the target cells and mixed therewith. A 100 μl aliquotthereof was added to a white plate to carry out the assay.

As shown in FIGS. 130A and 130B, hFR2-14_H1/L1, hFR2-14_H2/L1,hFR2-14_H3/L1, hFR2-14_H4/L1, and cFR2-14 had ADCC activity against theFGFR2 IIIb-expressing cells.

12)-2 ADCC Activity of Humanized Anti-FGFR2 Antibodies (hFR2-14_H3/L1,hFR2-14_H8/L1, and hFR2-14_H12/L1) Against FGFR2-Expressing Cancer CellLine

12)-2-1 Preparation of Target Cell

KATO III, NCI-H716, or SNU16 cells were washed twice with a medium forADCC and passed through a cell strainer (manufactured by BectonDickinson and Company). Then, the number of live cells was counted bythe trypan blue dye exclusion test. The cells were resuspended to 1×10⁵cells/ml and used as target cells.

12)-2-2 Preparation of PBMC Cell

25 ml of healthy human blood was gradually layered over 20 ml ofLymphosepar I (manufactured by Immuno-Biological Laboratories Co.,Ltd.), followed by centrifugation at 1500 rpm at room temperature for 30minutes. A cell layer located between plasma and Lymphosepar I(manufactured by Immuno-Biological Laboratories Co., Ltd.) was recoveredusing a dropper and suspended in 20 ml of a phenol red-free RPMI1640medium (manufactured by Life Technologies Corp.) containing 10% FBS. Thesuspension was centrifuged at 1500 rpm for 5 minutes. After removal ofthe supernatant, the cells were washed twice by the addition of 20 ml ofa medium for ADCC. The number of live cells was counted by the trypanblue dye exclusion test. After centrifugation, the medium was removed,and the cells were suspended in a medium for ADCC and used as effectorcells.

12)-2-3 Evaluation of ADCC Activity

The NCI-H716 cells prepared by the method of Example 12)-2-1 were addedat a concentration of 50 μl/well to a 96-well U-bottomed microplate.hFR2-14_H3/L1, hFR2-14_H8/L1, hFR2-14_H12/L1, or human IgG diluted to 1to 1000 ng/ml (final concentration) with a medium for ADCC was addedthereto at a concentration of 50 μl/well, and the plate was leftstanding at 4° C. for 1 hour. The PBMC cells (13.4×10⁶ cells/ml) ofExample 12)-2-2 were further added thereto at a concentration of 75μl/well. The plate was centrifuged at 1200 rpm at room temperature for 5minutes, followed by overnight culture at 37° C. under 5% CO₂conditions. On the next day, 10× Lysis Solution attached to CytoTox 96Non-Radioactive Cytotoxicity Assay (manufactured by Promega Corp.) kitwas added at a concentration of 17.5 μl/well to the wells containingonly the target cells and the medium and stirred, and the plate was thenleft standing at 37° C. for 45 minutes under 5% CO₂ conditions. Aftercentrifugation at 200 g at room temperature for 4 minutes, 50 μl of thesupernatant in each well was recovered into a 96-well flat-bottomedmicroplate (manufactured by Corning Inc.). Substrate Mix was addedthereto at a concentration of 50 μl/well, and the plate was leftstanding at room temperature for 30 minutes while shielded from light.Stop Solution was further added thereto at a concentration of 50μl/well. The absorbance was measured at 490 nm using a plate reader(ARVO; manufactured by PerkinElmer, Inc.). The percentage of cells lysedby ADCC activity was calculated according to the following expression:

Cytotoxicity(%)=(A−B−C)/(D−C)×100

A: Count of sample well

B: Average of spontaneous release (correction of culturesolution-containing wells from effector cell-containing wells) countsfrom effector cells (n=3).

C: Average of spontaneous release (correction of culturesolution-containing wells from target cell-containing wells) counts fromtarget cells (n=3).

D: Average of maximum release (correction of wells containing a culturesolution lysed in a surfactant from wells containing target cells lysedin a surfactant) counts (n=3).

As shown in FIG. 131, hFR2-14_H3/L1, hFR2-14_H8/L1, and hFR2-14_H12/L1had ADCC activity against the FGFR2-expressing cancer cell line NCI-H716cells.

12)-3 ADCC activity of humanized anti-FGFR2 antibodies (hFR2-14_H12/L1and hFR2-14_H19/L1) against FGFR2-expressing cancer cell line

The KATO III, NCI-H716, or SNU16 cells prepared by the method of Example12)-2-1 were added at a concentration of 50 μl/well to a 96-wellU-bottomed microplate. hFR2-14_H12/L1 or hFR2-14_H19/L1 (prepared inExamples 8) and 9)) or human IgG diluted to 1 to 1000 ng/ml (finalconcentration) with a medium for ADCC was added thereto at aconcentration of 50 μl/well, and the plate was left standing at 4° C.for 1 hour. The effector cells (20×10⁶ cells/ml) of Example 12)-2-2 werefurther added thereto at a concentration of 75 μl/well. The plate wascentrifuged at 1200 rpm at room temperature for 5 minutes, followed byovernight culture at 37° C. under 5% CO₂ conditions. On the next day,10× Lysis Solution attached to CytoTox 96 Non-Radioactive CytotoxicityAssay (manufactured by Promega Corp.) kit was added at a concentrationof 17.5 μl/well to the wells containing only the target cells and themedium and stirred, and the plate was then left standing at 37° C. for45 minutes under 5% CO₂ conditions. After centrifugation at 200 g atroom temperature for 4 minutes, 50 μl of the supernatant in each wellwas recovered into a 96-well flat-bottomed microplate (manufactured byCorning Inc.). Substrate Mix was added thereto at a concentration of 50μl/well, and the plate was left standing at room temperature for 30minutes while shielded from light. Stop Solution was further addedthereto at a concentration of 50 μl/well. The absorbance was measured at490 nm using a plate reader (ARVO; manufactured by PerkinElmer, Inc.).The percentage of cells lysed by ADCC activity was calculated accordingto the calculation method shown in Example 12)-2-3.

As shown in FIGS. 132A, 132B, and 132C, hFR2-14_H12/L1 andhFR2-14_H19/L1 had ADCC activity against the FGFR2-expressing cancercell lines NCI-H716, SNU-16, and KATO III, and this activity was shownto be higher in hFR2-14_H19/L1.

Example 13 ADCP Activity of Humanized Anti-Human FGFR2 Antibody(hFR2-14)

13)-1 ADCP Activity of Humanized Anti-FGFR2 Antibodies (hFR2-14_H12/L1and hFR2-14_H19/L1) Against FGFR2-Expressing Cancer Cell Line

13)-1-1 Preparation of Target Cell

KATO III or NCI-H716 cells were recovered and washed 3 times with PBS.Then, the number of live cells was counted by the trypan blue dyeexclusion test. 1×10⁶ cells were separated, centrifuged, and thensuspended in 200 μl of Diluent C attached to PKH26 Red Fluorescent CellLinker Kit for General Cell Membrane Labeling (manufactured bySigma-Aldrich Corp.). 1 mM PKH26 Linker was diluted as a labelingsolution to 10 μM with Diluent C. Immediately thereafter, the cellsuspension was mixed with an equal volume of the PKH26 Linker solution,and the mixture was left standing at room temperature for 5 minutes. Thecells were washed twice by the addition of 5 ml of an RPMI1640 medium(manufactured by Life Technologies Corp.) containing 10% FBS, thenresuspended to 5×10⁵ cells/ml, and used as target cells.

13)-1-2 Preparation of PBMC Cell

25 ml of healthy human blood was gradually layered over 20 ml ofLymphosepar I (manufactured by Immuno-Biological Laboratories Co.,Ltd.), followed by centrifugation at 1500 rpm at room temperature for 30minutes. A cell layer located between plasma and Lymphosepar I(manufactured by Immuno-Biological Laboratories Co., Ltd.) was recoveredusing a dropper and suspended in 20 ml of an RPMI1640 medium(manufactured by Life Technologies Corp.) containing 10% FBS. Thesuspension was centrifuged at 1500 rpm for 5 minutes. After removal ofthe supernatant, the cells were washed twice by the addition of 20 ml ofan RPMI1640 medium (manufactured by Life Technologies Corp.) containing10% FBS. The number of live cells was counted by the trypan blue dyeexclusion test. The resulting cells were used as effector cells.

13)-1-3 Preparation of Effector Cell

The PBMC cells prepared in Example 13)-1-2 was adjusted to 5×10⁷cells/ml with RoboSep buffer (manufactured by StemCell TechnologiesInc.). 50 μl of EasySep human Monocyte enrichment cocktail attached toHuman monocyte Enrichment Kit Without CD16 Depletion (manufactured byStemCell Technologies Inc.) was added per ml of the PBMC cellsuspension. After reaction at 4° C. for 10 minutes, 50 μl of EasySepMagnetic Particles was added per ml of the PBMC cell suspension. Afterreaction at 4° C. for 5 minutes, RoboSep buffer (manufactured byStemCell Technologies Inc.) was added thereto up to 2.5 ml, and thereaction mixture was loaded to EasySep Magnet. After 2 minutes and 30seconds, the supernatant was recovered and then centrifuged at 1200 rpmfor 5 minutes to separate monocyte fractions. The fractions were washedonce by the addition of an RPMI1640 medium (manufactured by LifeTechnologies Corp.) containing 10% FBS. Then, an RPMI1640 medium(manufactured by Life Technologies Corp.) containing 10% FBS, 10 ng/mlGM-CSF (manufactured by PeproTech Inc.), and 10 ng/ml M-CSF(manufactured by PeproTech Inc.) was added thereto, and the mixture wasinoculated to a 225-cm² flask for suspension culture (manufactured bySumitomo Bakelite Co., Ltd.). The cells were cultured at 37° C. for 14days under 5% CO₂ conditions. During the culture period, the medium wasreplaced every 3 to 4 days with an RPMI1640 medium (manufactured by LifeTechnologies Corp.) containing 10% FBS, 10 ng/ml GM-CSF (manufactured byPeproTech Inc.), and 10 ng/ml M-CSF (manufactured by PeproTech Inc.).Fourteen days later, 0.05% trypsin-EDTA (manufactured by LifeTechnologies Corp.) was added to macrophages differentiated therefrom byinduction. After reaction at 37° C. for 40 minutes, the cells weredissociated from the flask. The cells were washed twice by the additionof an RPMI1640 medium (manufactured by Life Technologies Corp.)containing 10% FBS, then resuspended to 5×10⁵ cells/ml in an RPMI1640medium (manufactured by Life Technologies Corp.) containing 10% FBS, 10ng/ml M-CSF (manufactured by PeproTech Inc.), and 250 U/ml IFN-γ(manufactured by PeproTech Inc.), and used as effector cells.

13)-1-4 Evaluation of ADCP Activity

The target cells prepared by the method of Example 13)-1-1 were added ata concentration of 100 μl/well to Ultra-Low Attachment 96-wellU-bottomed microplate (manufactured by Corning Inc.). hFR2-14_H12/L1 orhFR2-14_H19/L1 (prepared in Examples 8) and 9)) or human IgG diluted to0.5 to 500 ng/ml (final concentration) with an RPMI1640 medium(manufactured by Life Technologies Corp.) containing 10% FBS was addedthereto at a concentration of 100 μl/well, and the plate was leftstanding at 4° C. for 30 minutes. After centrifugation at 1200 rpm atroom temperature for 5 minutes and removal of the supernatant, the cellswere suspended in 100 μl/well of an RPMI1640 medium (manufactured byLife Technologies Corp.) containing 10% FBS. The effector cells (5×10⁵cells/ml) prepared in Example 13)-1-3 were added thereto at aconcentration of 100 μl/well, and the plate was then left standing at37° C. for 3 hours under 5% CO₂ conditions. After centrifugation at 1200rpm at 4° C. for 5 minutes and removal of the supernatant, the cellswere washed with 200 μl/well of PBS containing 5% FBS. 45 μl/well of PBScontaining 5% FBS and 5 μl/well of APC human CD11b (manufactured byBecton Dickinson and Company) were added to the cells, and the plate wasleft standing at 4° C. for 15 minutes. The cells were washed twice with200 μl/well of PBS containing 5% FBS. The cells were suspended in 200μl/well of PBS containing 1% paraformaldehyde, and the plate was leftovernight at 4° C. On the next day, the cells were assayed by flowcytometry (FACS Canto II; manufactured by Becton Dickinson and Company).The data was analyzed using Flowjo (manufactured by Tree Star Inc.).After development on FSC (forward scatter)/SSC (side scatter), thenumber of PE-positive cells (A) and the number of cells positive forboth APC and PE (B) were calculated. The cells positive for both APC andPE (B) mean that the macrophages englobed the target cells. Thepercentage of cells phagocytosed by ADCP activity was calculatedaccording to the following expression:

Percentage of cells phagocytosed(%)=B/(A+B)×100

As shown in FIGS. 133A and 133B, hFR2-14_H12/L1 and hFR2-14_H19/L1 hadADCP activity against the FGFR2-expressing cancer cell lines NCI-H716and KATO III.

Example 14 In Vivo Antitumor Activity of Humanized Anti-FGFR2 Antibody(hFR2-14)

14)-1 In Vivo Antitumor Activity of Humanized Anti-FGFR2 Antibodies(hFR2-14_H1/L1, hFR2-14_H2/L1, hFR2-14_H3/L1, and hFR2-14_H4/L1) AgainstHuman Stomach Cancer Cell Line SNU-16-Subcutaneously Transplanted Model

5×10⁶ cells of a human stomach cancer line SNU-16 (purchased from ATCC)were suspended in 50% Matrigel (purchased from Nippon Becton DickinsonCompany, Ltd.) and subcutaneously transplanted to the axillary region ofeach nude mouse (CAnN.Cg-Foxnl^(nu)/CrlCrlj, purchased from CharlesRiver Laboratories Japan Inc.). The mice were grouped according to theirtumor volumes. Seven, 10, 14, 17, and 21 days after transplantation,each humanized anti-FGFR2 antibody (hFR2-14_H1/L1, hFR2-14_H2/L1,hFR2-14_H3/L1, or hFR2-14_H4/L1) prepared in Example 8)-3 wasintraperitoneally administered at a dose of 1.5 or 15 mg/kg to thecancer-bearing mice (n=8). The major axis and minor axis of thetransplanted tumor were measured twice a week using an electronicdigital caliper (manufactured by Mitsutoyo Corp.). The tumor volume wascalculated according to the following expression:

Tumor volume(mm³)=½×Minor axis(mm)×Minor axis(mm)×Major axis(mm)

The results for the hFR2-14_H1/L1 antibody are shown in FIG. 134A. Thepercentage of tumor growth inhibition at 21 days after transplantation(final assay day) was 58% for the 1.5 mg/kg administration group and 53%for the 15 mg/kg administration group.

The results for the hFR2-14_H2/L1 antibody are shown in FIG. 134B. Thepercentage of tumor growth inhibition at 21 days after transplantation(final assay day) was 58% for the 1.5 mg/kg administration group and 58%for the 15 mg/kg administration group.

The results for the hFR2-14_H3/L1 antibody are shown in FIG. 134C. Thepercentage of tumor growth inhibition at 21 days after transplantation(final assay day) was 64% for the 1.5 mg/kg administration group and 41%for the 15 mg/kg administration group.

The results for the hFR2-14_H4/L1 antibody are shown in FIG. 134D. Thepercentage of tumor growth inhibition at 21 days after transplantation(final assay day) was 70% for the 1.5 mg/kg administration group and 39%for the 15 mg/kg administration group.

14)-2 In Vivo Antitumor Activity of Humanized Anti-FGFR2 Antibodies(hFR2-14_H5/L1, hFR2-14_H8/L1, hFR2-14_H9/L1, hFR2-14_H11/L1,hFR2-14_H12/L1, and hFR2-14_H19/L1) Against Human Stomach Cancer CellLine SNU-16-Subcutaneously Transplanted Model

5×10⁶ cells of a human stomach cancer line SNU-16 (purchased from ATCC)were suspended in 50% Matrigel (purchased from Nippon Becton DickinsonCompany, Ltd.) and subcutaneously transplanted to the axillary region ofeach nude mouse (CAnN.Cg-Foxnl^(nu)/CrlCrlj, purchased from CharlesRiver Laboratories Japan Inc.). The mice were grouped according to theirtumor volumes. Seven, 10 (or 11), 14, and 17 (or 18) days aftertransplantation, each humanized antibody (hFR2-14_H5/L1, hFR2-14_H8/L1,hFR2-14_H9/L1, hFR2-14_H11/L1, or hFR2-14_H12/L1) prepared in Example8)-3 or the humanized antibody (hFR2-14_H19/L1) prepared in Example 9)was intraperitoneally administered at a dose of 2 or 20 mg/kg to thecancer-bearing mice (n=8 or 9). The major axis and minor axis of thetransplanted tumor were measured twice a week using an electronicdigital caliper (manufactured by Mitsutoyo Corp.). The tumor volume wascalculated according to the following expression:

Tumor volume(mm³)=½×Minor axis(mm)×Minor axis(mm)×Major axis(mm)

The results on hFR2-14_H5/L1 are shown in FIG. 135A. The percentage oftumor growth inhibition at 21 days after transplantation (final assayday) was 41% for the 2 mg/kg administration group and 51% for the 20mg/kg administration group.

The results for hFR2-14_H8/L1 are shown in FIG. 135B. The percentage oftumor growth inhibition at 21 days after transplantation (final assayday) was 30% for the 2 mg/kg administration group and 30% for the 20mg/kg administration group.

The results for hFR2-14_H9/L1 are shown in FIG. 135C. The percentage oftumor growth inhibition at 21 days after transplantation (final assayday) was 1% for the 2 mg/kg administration group and 23% for the 20mg/kg administration group.

The results for hFR2-14_H11/L1 are shown in FIG. 135D. The percentage oftumor growth inhibition at 21 days after transplantation (final assayday) was 61% for the 2 mg/kg administration group and 42% for the 20mg/kg administration group.

The results for hFR2-14_H12/L1 are shown in FIG. 135E. The percentage oftumor growth inhibition at 21 days after transplantation (final assayday) was 36% for the 2 mg/kg administration group and 58% for the 20mg/kg administration group.

The results for hFR2-14_H19/L1 are shown in FIG. 135F. The percentage oftumor growth inhibition at 21 days after transplantation (final assayday) was 70% for the 2 mg/kg administration group and 40% for the 20mg/kg administration group.

14)-3 In Vivo Antitumor Activity of Humanized Anti-FGFR2 Antibodies(hFR2-14_H12/L1 and hFR2-14_H19/L1) Against Human Colorectal Cancer CellLine NCI-H716 Block-Transplanted Model

A tumor block (5×5×5 mm³) of a human colorectal cancer line NCI-H716(purchased from ATCC) was subcutaneously transplanted to the axillaryregion of each nude mouse (CAnN.Cg-Foxnl^(nu)/CrlCrlj, purchased fromCharles River Laboratories Japan Inc.). On the transplantation day and3, 7, 10, 14, 17, 21, and 24 days after transplantation, each humanizedantibody (hFR2-14_H12/L1 or hFR2-14_H19/L1) prepared in Example 8) or 9)was intraperitoneally administered at a dose of 20 mg/kg to thecancer-bearing mice (n=11). The major axis and minor axis of thetransplanted tumor were measured twice a week using an electronicdigital caliper (manufactured by Mitsutoyo Corp.). The tumor volume wascalculated according to the following expression:

Tumor volume(mm³)=½×Minor axis(mm)×Minor axis(mm)×Major axis(mm)

The results for hFR2-14_H12/L1 are shown in FIG. 136A. The percentage oftumor growth inhibition at 28 days after transplantation (final assayday) was 99%.

The results for hFR2-14_H19/L1 are shown in FIG. 136B. The percentage oftumor growth inhibition at 28 days after transplantation (final assayday) was 99%.

14)-4 In Vivo Antitumor Activity of Humanized Anti-FGFR2 Antibodies(hFR2-14_H12/L1 and hFR2-14_H19/L1) Against Human Colorectal Cancer CellLine NCI-H716-Luc-Peritoneally Disseminated Model

3×10⁶ cells of luciferase gene-expressing human colorectal cancer lineNCI-H716-luc (NCI-H716 (purchased from ATCC) transfected with theluciferase gene) were suspended in saline (purchased from OtsukaPharmaceutical Co., Ltd.) and intraperitoneally transplanted to each NOGmouse (NOD/Shi-SCID, IL-2Rγnull, purchased from Central Institute forExperimental Animals). One day after transplantation, the mice weregrouped according to their weights. One, 5, 8, 12, 16, 19, and 26 daysafter transplantation, each humanized antibody (hFR2-14_H12/L1 orhFR2-14_H19/L1) prepared in Examples 8) and 9) was intraperitoneallyadministered at a dose of 20 mg/kg to the cancer-bearing mice (n=11).The date of death of a mouse was recorded, and the survival rate (%) wascalculated. Thirty-seven days after transplantation, VivoGlo Luciferin(purchased from Promega Corp.) was administered at a dose of 150 mg/kgto the tail veins of the cancer-bearing mice. Ten minutes afteradministration, the luciferase activity of the cancer-bearing mice wasassayed using IVIS (manufactured by Caliper, A PerkinElmer Company). Theluciferase activity (p/s/cm²/sr) was quantified using Living Image(manufactured by Caliper, A PerkinElmer Company).

FIG. 137(A) shows the tumor growth inhibitory effects of the humanizedantibodies. The percentage of tumor growth inhibition relative to thenon-administered group, with the luciferase activity as an index, at 37days after transplantation was 98% for the hFR2-14_H12/L1-administeredgroup and 98% for the hFR2-14_H19/L1-administered group.

FIG. 137(B) shows the life prolonging effects of the humanizedantibodies. The survival rate at 93 days after transplantation was 0%for the non-administered group, whereas the survival rate was 64% forthe hFR2-14_H12/L1 antibody-administered group and 91% for thehFR2-14_H19/L1 antibody-administered group.

Example 15 X-Ray Structural Analysis of Complex of Humanized Anti-FGFR2Antibody (hFR2-14_H3/L1) and FGFR2 Protein

15)-1 Preparation of FGFR2 Protein for Crystallization

15)-1-1 Preparation of FGFR2 Protein Expression Vector forCrystallization

In order to construct a vector for expression of a region consisting ofan amino acid sequence of amino acid positions 148 to 249 in the commonportion of human FGFR2 IIIb and IIIc (FIG. 78; SEQ ID NO: 70 and FIG.79; SEQ ID NO: 71) (hereinafter, this region is referred to as “D2”),PCR reaction was performed using a primer set shown below and a vectorplasmid comprising amino acid positions 126 to 313 of human FGFR2 (FIG.78; SEQ ID NO: 70 and FIG. 79; SEQ ID NO: 71) as a template.

Primer Set for Gene Amplification of D2:

D23fw: (FIG. 120; SEQ ID NO: 112)5′-CTGTTTCAAGGTCCGAGCAATAACAAACGTGCACCGTATTGG-3′ and D23rv:(FIG. 121; SEQ ID NO: 113)5′-CGCAAGCTTGTCGACTCAAACAACATCCAGATGATAGGTATG-3′.The obtained PCR product was inserted to pET24b(+) (manufactured byMerck KGaA (Novagen)) preliminarily containing nucleotide sequencesencoding a His tag and an HRV3c protease cleavage site using In-FusionHD Cloning Kit (manufactured by Takara Bio Inc.) (hereinafter, theresulting vector is referred to as “pET24b(+)-D2”, and hereinafter andin the drawings, the recombinant protein expressed by “pET24b(+)-D2” isreferred to as FGFR2D2).

15)-1-2 Preparation of FGFR2 Protein (FGFR2D2) for Crystallization

E. coli Origami 2 (DE3) (manufactured by Merck KGaA (Novagen)) wastransformed with the expression plasmid pET24b(+)-D2 and preculturedovernight at 200 rpm at 37° C. in 50 mL of Terrific medium (manufacturedby ForMedium Ltd.) supplemented with 25 μg/ml kanamycin (manufactured byWako Pure Chemicals Industries, Ltd.) and 12 μg/ml tetracycline(manufactured by Wako Pure Chemicals Industries, Ltd.). 50 mL of theprecultured solution was added to 1 L of Terrific medium alsosupplemented with 25 μg/ml kanamycin and 12 μg/ml tetracycline, andcultured at 250 rpm at 37° C. for 1 hour. Then, the temperature waslowered to 16° C., and the expression of FGFR2D2 was induced by theaddition of 1 mM IPTG, followed by culture for 26 hours. The bacterialcells were collected by centrifugation at 4500 rpm for 30 minutes,suspended in a binding buffer (50 mM Tris-HCl (pH 8.0), 400 mM NaCl, and20 mM imidazole) containing Inhibitor Cocktail (manufactured by RocheApplied Science) dissolved therein, and then sonicated on ice. Aftercentrifugation at 25000 rpm for 20 minutes, the supernatant wasrecovered and applied to HisTrap FF crude column (manufactured by GEHealthcare Bio-Sciences Corp.). The column was washed with the bindingbuffer, followed by gradient elution with an elution buffer (50 mMTris-HCl (pH 8.0), 400 mM NaCl, and 500 mM imidazole) to collectfractions containing the protein of interest. The collected sample wasdiluted with a buffer (50 mM Tris-HCl (pH 7.5) and 0.1 mM EDTA) and thenapplied to HiTrap SP HP, followed by gradient elution with a buffer (50mM Tris-HCl (pH 7.5), 1 M NaCl, and 0.1 mM EDTA) to collect fractionscontaining the protein of interest. The obtained sample was applied to agel filtration column (HiLoad 16/600 Superdex 75 pg; manufactured by GEHealthcare Bio-Sciences Corp.) equilibrated with a buffer (25 mM HEPES(pH 7.5), 300 mM NaCl, and 0.1 mM EDTA) to collect fractions containingthe protein of interest. The obtained FGFR2D2 was concentrated into 15mg/mL using Amicon Ultra-4 (manufactured by Merck Millipore).

15)-2 Preparation of Fab Fragment of Humanized Anti-FGFR2 Antibody(hFR2-14_H3/L1)

hFR2-14_H3/L1 was dialyzed against 20 mM phosphate and 10 mM EDTA (pH7.0) and then concentrated into 24 mg/ml using Amicon Ultra-15 MWCO 10K(manufactured by Merck Millipore) to prepare 1.9 ml of a concentrate.5.5 ml of cysteine (manufactured by Sigma-Aldrich Corp.) adjusted to0.005 mM with 20 mM phosphate and 10 mM EDTA (pH 7.0), and 0.19 ml ofpapain (manufactured by Sigma-Aldrich Corp.) diluted 1/100 with 20 mMphosphate and 10 mM EDTA (pH 7.0) were added to the concentrate andreacted at 37° C. for 18 hours. After 18 hours, the reaction was stoppedby the addition of 2.53 ml of N-ethylmaleimide (manufactured by TokyoChemical Industry Co., Ltd.) dissolved at a concentration of 120 mM in20 mM phosphate and 10 mM EDTA (pH 7.0). The reaction solution wasapplied to MabSelect SuRe 5 ml (manufactured by GE HealthcareBio-Sciences Corp.) equilibrated with PBS to recover 18 ml of aflow-through fraction corresponding to the Fab fragment. The fractionwas concentrated using Amicon Ultra-15 MWCO 10K (manufactured by MerckMillipore) and applied to Superdex 200 16/60 (manufactured by GEHealthcare Bio-Sciences Corp.) equilibrated with 50 mM Tris-HCl and 20mM NaCl (pH 7.5) to recover 18 ml of a fraction corresponding to the Fabfragment (hereinafter, the resulting fragment is referred to as an“H3L1Fab fragment”).

15)-3 Preparation of H3L1Fab Fragment/FGFR2D2 Complex Sample

0.5 mL of 15 mg/mL FGFR2D2 was mixed per mL of 12.5 mg/mL H3L1Fabfragment, and the mixture was left overnight at 4° C. and then appliedto HisTrap FF crude equilibrated with a binding buffer (50 mM Tris-HCl(pH 8.0), 400 mM NaCl, and 20 mM imidazole). The column was washed withthe binding buffer, followed by elution with an elution buffer (50 mMTris-HCl (pH 8.0), 400 mM NaCl, and 500 mM imidazole). The eluted samplewas applied to a gel filtration column (HiLoad 16/600 Superdex 200 pg;manufactured by GE Healthcare Bio-Sciences Corp.) equilibrated with abuffer (25 mM Tris-HCl (pH 7.5) and 50 mM NaCl) to recover 11 mL of afraction corresponding to the complex.

15)-4 Crystallization and Structural Analysis of H3L1FabFragment/FGFR2D2 Complex

The obtained complex of H3L1Fab and FGFR2D2 was concentrated into 22mg/mL and used in crystallization. The vapor diffusion method was usedfor the crystallization. To 0.5 to 0.7 μL of the protein solution, anequal amount of a precipitant solution (1.225 M ammonium sulfate and0.15 M Tris-HCl, pH 8.5) was added, and the resulting solution wasplaced in a sealed container containing 0.45 mL of a precipitantsolution such that these solutions had no contact with each other. Thecontainer was left standing at 20° C. Three days later, 0.2 mm×0.2mm×0.2 mm single crystals were obtained.

The obtained crystals were dipped in a precipitant solution supplementedwith 30% (v/v) ethylene glycol and subsequently frozen under nitrogenstream of −180° C. X-ray diffraction data was collected under nitrogenstream of 95 K using BL5A of Photon Factory, Institute of MaterialsStructure Science, High Energy Accelerator Research Organization.Diffraction intensity was digitized from the obtained diffraction imageusing software HKL2000 (manufactured by HKL Research Inc.) to determinecrystal structure factors. The crystals were in the tetragonal systemwith a space group of P41212 and unit cells of a=b=60.57 angstroms andc=331.2 angstroms.

The molecular replacement method was performed using the obtainedstructure factors and the three-dimensional structure coordinates ofFGFR2 (the portion concerned was extracted from PDB code: 3OJ2) and Fab(the antibody structure previously subjected to crystal structuralanalysis was utilized) to determine a phase. Software phaser (CCP4:Collaborative Computational Project No. 4) was used in calculation. Thecrystals each contained 1 complex in the asymmetric unit.

Structure refinement was performed using software refmac5 (CCP4), andmodel correction was performed using software coot. This operation wasrepetitively performed to obtain a final R factor of 21.8% and a free Rfactor of 25.8% with a resolution of 2.3 angstroms. The model iscomposed of 1 complex and contains amino acid residues 1 to 215 of theH3L1Fab L chain, amino acid residues 1 to 221 of the H3L1Fab H chain,amino acid residues 150 to 249 of FGFR2D2, 1 sulfate ion, and 285 watermolecules. The C-terminal 1 residue of the H3L1Fab L chain, theC-terminal 4 residues of the H3L1Fab H chain, and N-terminal 20 residuescomprising the His tag and protease cleavage site of FGFR2D2 were notincluded in the model because of their obscure electric density.

The determined amino acid residues of FGFR2D2 within 4 angstroms fromH3L1Fab are as follows: Tyr155, Thr157, Lys176, Ala181, Gly182, Gly183,Asn184, Pro185, Met186, Thr188, Gln200, Glu201, Gly205, Gly206, Lys208,Val209, Arg210, Asn211, Gln212, His213, Trp214, and Ile217. FIG. 138shows a ribbon model of the whole complex. FIG. 139 shows a diagramshowing the superposition of the D2 region in the FGFR2D2/H3L1Fabcomplex onto the corresponding region of an FGFR2/FGF1 complex structure(PDB code: 3OJ2).

Example 16 Immunostaining Using Rat Anti-FGFR2 Antibody FR2-10

16)-1 Preparation of Sample for Immunostaining

16)-1-1 Preparation of Cell Line Expressing Each Molecule of FGFR Family

293α cells (described in Example 1)-6) were adjusted to 6×10⁶cells/225-cm² flask (manufactured by Sumitomo Bakelite Co., Ltd.) in aDMEM medium containing 10% FBS and cultured overnight at 37° C. under 5%CO₂ conditions. The cells were transfected with the FGFR1 IIIb, FGFR1IIIc, FGFR2 IIIb, FGFR2 IIIc, FGFR3 IIIb, FGFR3 IIIc, or FGFR4expression vector constructed in Examples 1)-3-1, 2)-1-1, and 10)-2-1 oran empty vector, i.e., pcDNA-DEST04-FGFR1 IIIb, pcDNA-DEST40-FGFR1 IIIc,pcDNA-DEST40-FGFR2 IIIb, pcDNA-DEST40-FGFR2 IIIc, pcDNA-DEST40-FGFR3IIIb, pcDNA-DEST40-FGFR3 IIIc, pcDNA-DEST40-FGFR4, or pcDNA-DEST40 usingFuGENE 6 (manufactured by Roche Diagnostics K.K.) and cultured for twonights at 37° C. under 5% CO₂ conditions. The obtained cells wererecovered using TrypLE Express (manufactured by Life Technologies Corp.)and centrifuged to obtain a pellet, which was then washed once with PBSand centrifuged. The resulting pellet was fixed in 20% neutral bufferedformalin.

16)-1-2 Preparation of FGFR2-Expressing Cancer Cell Line

A human stomach cancer line SNU-16 and a human colorectal cancer lineNCI-H716 (purchased from ATCC) cultured in RPMI containing 10% FBS wereeach recovered and centrifuged to obtain pellet, which was then fixed in20% neutral buffered formalin. A human stomach cancer line KATO III(purchased from ATCC) cultured in DMEM containing 10% FBS was recoveredand centrifuged to obtain a pellet, which was then fixed in 20% neutralbuffered formalin.

16)-1-3 Preparation of Tumor Sample of FGFR2-Expressing Cancer Cell LineXenograft Model

5×10⁶ cells of SNU-16 were suspended in 50% Matrigel (manufactured byNippon Becton Dickinson Company, Ltd.) and subcutaneously transplantedto the axillary region of each nude mice (CAnN.Cg-Foxnl^(nu)/CrlCrlj,purchased from Charles River Laboratories Japan Inc.). Twenty days aftertransplantation, tumor was recovered and fixed in Mildform (manufacturedby Wako Pure Chemicals Industries, Ltd.).

3×10⁵ cells of KATO III were suspended in 100% Matrigel (manufactured byNippon Becton Dickinson Company, Ltd.) and subcutaneously transplantedto the axillary region of each SCID mouse(CB17/lcr-Prkdc^(scid)/CrlCrlj, purchased from Charles RiverLaboratories Japan Inc.). Thirty days after transplantation, tumor wasrecovered and fixed in Mildform (manufactured by Wako Pure ChemicalsIndustries, Ltd.).

2.5×10⁶ cells of NCI-H716 were suspended in 100% Matrigel (manufacturedby Nippon Becton Dickinson Company, Ltd.) and subcutaneouslytransplanted to the axillary region of each nude mice(CAnN.Cg-Foxnl^(nu)/CrlCrlj, purchased from Charles River LaboratoriesJapan Inc.). Twenty-one days after transplantation, tumor was recovered.

16)-2 Paraffin Embedding and Sectioning

Paraffin embedding and sectioning are general approaches, and any toolor instrument can be used without particular limitations.

The cells of each line prepared in Examples 16)-1-1 and 16)-1-2 wererecovered into a 15-mL tube and centrifuged at 1500 rpm for 5 minutes toremove a supernatant. 3 mL of 20% neutral buffered formalin(manufactured by Wako Pure Chemicals Industries, Ltd.) was layered overthe cell pellet and left standing at room temperature for 30 minutes orlonger for fixation. Then, 5 mL of chloroform was added thereto.Immediately thereafter, the tube was centrifuged at 1000 rpm for 10minutes, and the formalin layer was immediately removed. Then, the cellpellet formed between the formalin layer and the chloroform layer wasrecovered. The cell pellet was put in a nylon mesh bag, which was thenplaced in a cassette for tissue preparation (Unicassette Standard,manufactured by Sakura Finetek Japan Co., Ltd.). The cell pellet,together with the cassette, was dipped in ethanol to wash off thechloroform. Each xenograft tissue prepared in Example 16)-1-3 was fixedin Mildform (purchased from Wako Pure Chemicals Industries, Ltd.), thentrimmed at the cutout portion, and placed in a cassette.

The cell pellet and the xenograft tissue were paraffin-embedded by aconventional method. Dehydration, delipidation, and paraffinimpregnation were performed using an automatic fixation and embeddingapparatus (Tissue-Tek VIP5 Jr.; manufactured by Sakura Finetek JapanCo., Ltd.). The cassette was taken out of the automatic fixation andembedding apparatus and transferred to the paraffin bath of aparaffin-embedded block preparation apparatus (Tissue-Tek TEC Plus;manufactured by Sakura Finetek Japan Co., Ltd.). A small amount ofmelted paraffin was injected into an embedding dish loaded to thisapparatus. The cell pellet or the tissue was separated with tweezersfrom the cassette container or the nylon mesh taken out of the paraffinbath, and loaded into the paraffin in this embedding dish. Subsequently,a cassette was placed as an embedding frame on the embedding dish, andmelted paraffin was poured over the cell pellet or the tissue within thecassette. The embedding dish containing the embedding frame integratedwith the cells or the tissue was placed on a cooling unit and cooled.After solidification of paraffin, the embedded block was taken out ofthe embedding dish and subjected to sectioning. The sectioning wasperformed by the slicing of the embedded block thus prepared intosections with a thickness of 3 μm using a microtome (IVS-410;manufactured by Sakura Finetek Japan Co., Ltd.). Each section thusobtained was applied to an antistripping glass slide (Platinum;manufactured by Matsunami Glass Ind., Ltd.). The glass slide was driedovernight on a paraffin stretcher (manufactured by Sakura Finetek JapanCo., Ltd.) at 50° C., accommodated in a slide case, and stored in adesiccator.

16)-3 Staining

Each sample was stained using an automatic staining apparatus (DiscoveryUltra; manufactured by Ventana Medical Systems, Inc.). The reactiontemperature during the staining process was set to 37° C., unlessotherwise specified. The amounts of various reagents added were all setto one drop. The sample was deparaffinized by 3 incubation runs eachinvolving 68° C. for 4 minutes using fresh EZ buffer (manufactured byVentana Medical Systems, Inc.). The sample was washed with EZ buffer.Cell conditioning was carried out by 4 runs each involving 95° C. usingfresh CC1 buffer (manufactured by Ventana Medical Systems, Inc.) (atotal of 52 minutes). The sample was washed 4 times with a reactionbuffer (manufactured by Ventana Medical Systems, Inc.). The ratanti-FGFR2 antibody FR2-10 and a commercially available antibodyAnti-Human K-sam Rabbit IgG Affinity Purify (manufactured by IBL Co.,Ltd.) were diluted to 10 μg/mL and 210 μg/mL, respectively, with anantibody diluent dedicated to Discovery (manufactured by Ventana MedicalSystems, Inc.), and reacted with the sample for 1 hour. After washing 4times with a reaction buffer, a solution of biotinylated goat anti-ratIgG (manufactured by Jackson ImmunoResearch Laboratories, Inc.) diluted500-fold with an antibody diluent dedicated to Discovery and DiscoveryUniversal Secondary Antibody (manufactured by Ventana Medical Systems,Inc.) were reacted therewith for 32 minutes. The sample was washed twicewith a reaction buffer. Inhibitor D (DAB Map kit; manufactured byVentana Medical Systems, Inc.) was reacted therewith for 8 minutes, andthe sample was then washed twice with a reaction buffer. SA-HRP D (DABMap kit; manufactured by Ventana Medical Systems, Inc.) was reactedtherewith for 16 minutes, and the sample was washed twice with areaction buffer. DAB D (DAB Map kit; manufactured by Ventana MedicalSystems, Inc.) was reacted therewith for 4 minutes. Then, DAB H₂O₂ (DABMap kit; manufactured by Ventana Medical Systems, Inc.) was addedthereto, followed by reaction with 8 minutes. The sample was washedtwice with a reaction buffer. Copper-D (DAB Map kit; manufactured byVentana Medical Systems, Inc.) was reacted therewith for 4 minutes, andthe sample was washed 3 times with a reaction buffer. Hematoxylinnuclear staining reagent II (manufactured by Ventana Medical Systems,Inc.) was reacted therewith for 4 minutes, and the sample was washedtwice with a reaction buffer. A lithium carbonate reagent (manufacturedby Ventana Medical Systems, Inc.) was reacted therewith for 4 minutes,and the sample was washed once with a reaction buffer.

The completely stained preparations were dehydrated with ethanol series,cleared with xylene series, and then mounted on glass covers togetherwith mounting agents. The preparations were observed under an opticalmicroscope and evaluated for brown stains representing positive reactionproducts.

As shown in FIG. 140, the rat anti-FGFR2 antibody FR2-10 exhibited verystrong stains only on some cells in the blocks of cells forced toexpress FGFR2 IIIb. No positive stain was observed in other forcedlyexpressing cells or empty vector-transfected cells. Thus, it wasconcluded that the rat anti-FGFR2 antibody FR2-10 is capable ofspecifically staining FGFR2 IIIb.

As shown in FIG. 141, the commercially available anti-FGFR2 antibodyexhibited clear positive stains on many cells in the blocks of SNU-16cells (FIG. 141-D), KATO III cells (FIG. 141-E), and NCI-H716 cells(FIG. 141-F), demonstrating that these cell lines expressed the FGFR2protein. On the other hand, the rat anti-FGFR2 antibody FR2-10 exhibitedclear positive stains on many cells in the blocks of SNU-16 cells (FIG.141-A) and KATO III cells (FIG. 141-B), but exhibited no positive stainon the NCI-H716 cells (FIG. 141-C), demonstrating that the NCI-H716cells did not express the FGFR2 IIIb protein.

As shown in FIG. 142, the rat anti-FGFR2 antibody FR2-10 exhibited clearpositive stains on many cells in the xenograft tumors derived fromSNU-16 cells (FIG. 142-A) and KATO III cells (FIG. 142-B), but exhibitedno stain on the xenograft tumor derived from NCI-H716 cells (FIG.142-C).

Example 17 Inhibitory Activity of Humanized Anti-Human FGFR2 Antibody(hFR2-14) Against Ligand-Receptor Binding

The binding of the ligand FGF7 to the antigen (C-terminally His-taggedrhFGFR2 alpha (IIIb)) was detected by ELISA. rhFGFR2 alpha (IIIb) wasdiluted to 2 μg/ml with PBS and added at a concentration of 100 μl/wellto 96 well Clear Polystyrene High Bind Stripwell Microplate(manufactured by Corning Inc.), and the plate was left overnight at 4°C. On the next day, the solution was removed using an aspirator, and thecontents in the wells were washed 3 times with PBS containing 0.05%Tween-20 (manufactured by Bio-Rad Laboratories, Inc.). Then, PBScontaining 1% BSA (manufactured by Sigma-Aldrich Corp.) was addedthereto at a concentration of 200 μl/well, and the plate was left atroom temperature for 1 hour. After removal of the solution, the contentsin the wells were washed 3 times with PBS containing 0.05% Tween-20.FGF7 diluted to 9 ng/ml with PBS containing 1% BSA, heparin(manufactured by Sigma-Aldrich Corp.) diluted to 300 μg/ml, and thecFR2-10 (prepared in Example 4)), hFR2-14_H12/L1 (prepared in Example8)), or hFR2-14_H19/L1 antibody (prepared in Example 9)) diluted to 0.3to 30 μg/ml were each added thereto at a concentration of 50 μl/well,and the plate was left at room temperature for 2 hours. After removal ofthe solution, the contents in the wells were washed 3 times with PBScontaining 0.05% Tween-20. A biotinylated anti-FGF-7 antibody (HumanKGF/FGF-7 DuoSet, manufactured by R&D Systems, Inc.) diluted 180-foldwith PBS containing 1% BSA was added thereto at a concentration of 100μl/well, and the plate was left at room temperature for 2 hours. Afterremoval of the solution, the contents in the wells were washed 3 timeswith PBS containing 0.05% Tween-20. Streptavidin-HRP (Human KGF/FGF-7DuoSet, manufactured by R&D Systems, Inc.) diluted 200-fold with PBScontaining 1% BSA was added thereto at a concentration of 100 μl/well,and the plate was left at room temperature for 20 minutes while shieldedfrom light. After removal of the solution, the contents in the wellswere washed 3 times with PBS containing 0.05% Tween-20. A substratesolution containing Reagent A and Reagent B mixed in equal amounts(Substrate Reagent Pack, manufactured by R&D Systems, Inc.) was addedthereto at a concentration of 100 μl/well, and the plate was left atroom temperature for 20 minutes for color reaction while shielded fromlight. The reaction was stopped by the addition of Stop solution(manufactured by R&D Systems, Inc.) at a concentration of 50 μl/well.Then, the absorbance was measured at 450 nm and 570 nm using a platereader. The measurement value at 570 nm was subtracted from themeasurement value at 450 nm to determine a value. As shown in FIG. 143,the cFR2-10, hFR2-14_H12/L1, and hFR2-14_H19/L1 antibodies all had theactivity of inhibiting the binding of the ligand to the receptor.

INDUSTRIAL APPLICABILITY

Use of the antibody provided by the present invention enables treatmentor prevention of various cancers and testing or diagnosis of variouscancers.

Sequence Listing Free Text

SEQ ID NO: 1: N-terminal amino acid sequence of a band corresponding tothe heavy chain of a rat anti-FGFR2 antibody FR2-10 (FIG. 9).SEQ ID NO: 2: N-terminal amino acid sequence of a band corresponding tothe light chain of a rat anti-FGFR2 antibody FR2-10 (FIG. 10).SEQ ID NO: 3: N-terminal amino acid sequence of a band corresponding tothe heavy chain of a rat anti-FGFR2 antibody FR2-13 (FIG. 11).SEQ ID NO: 4: N-terminal amino acid sequence of a band corresponding tothe light chain of a rat anti-FGFR2 antibody FR2-13 (FIG. 12).SEQ ID NO: 5: N-terminal amino acid sequence of a band corresponding tothe heavy chain of a rat anti-FGFR2 antibody FR2-14 (FIG. 13).SEQ ID NO: 6: N-terminal amino acid sequence of a band corresponding tothe light chain of a rat anti-FGFR2 antibody FR2-14 (FIG. 14).SEQ ID NO: 7: Primer for gene amplification of a rat heavy chain (FIG.15).SEQ ID NO: 8: Sequencing primer for the heavy chain of FR2-10 (FIG. 16).SEQ ID NO: 9: Sequencing primer for the heavy chain of FR2-13 (FIG. 17).SEQ ID NO: 10: Sequencing primer for the heavy chain of FR2-14 (FIG.18).SEQ ID NO: 11: Nucleotide sequence of a cDNA encoding the heavy chainvariable region of a rat anti-FGFR2 antibody FR2-10 (FIG. 19).SEQ ID NO: 12: Amino acid sequence of the heavy chain variable region ofthe rat anti-FGFR2 antibody FR2-10 (FIG. 20).SEQ ID NO: 13: Nucleotide sequence of a cDNA encoding the heavy chainvariable region of a rat anti-FGFR2 antibody FR2-13 (FIG. 21).SEQ ID NO: 14: Amino acid sequence of the heavy chain variable region ofthe rat anti-FGFR2 antibody FR2-13 (FIG. 22).SEQ ID NO: 15: Nucleotide sequence of a cDNA encoding the heavy chainvariable region of a rat anti-FGFR2 antibody FR2-14 (FIG. 23).SEQ ID NO: 16: Amino acid sequence of the heavy chain variable region ofthe rat anti-FGFR2 antibody FR2-14 (FIG. 24).SEQ ID NO: 17: Primer for gene amplification of a rat light chain (FIG.25).SEQ ID NO: 18: Sequencing primer for a rat light chain (FIG. 26).SEQ ID NO: 19: Sequencing primer for the light chain of FR2-10 (FIG.27).SEQ ID NO: 20: Nucleotide sequence of a cDNA encoding the light chainvariable region of a rat anti-FGFR2 antibody FR2-10 (FIG. 28).SEQ ID NO: 21: Amino acid sequence of the light chain variable region ofthe rat anti-FGFR2 antibody FR2-10 (FIG. 29).SEQ ID NO: 22: Primer for gene amplification of the rat FR2-13 or FR2-14light chain (FIG. 30).SEQ ID NO: 23: Nucleotide sequence of a cDNA encoding the light chainvariable region of a rat anti-FGFR2 antibody FR2-13 (FIG. 31).SEQ ID NO: 24: Amino acid sequence of the light chain variable region ofthe rat anti-FGFR2 antibody FR2-13 (FIG. 32).SEQ ID NO: 25: Nucleotide sequence of a cDNA encoding the light chainvariable region of a rat anti-FGFR2 antibody FR2-14 (FIG. 33).SEQ ID NO: 26: Amino acid sequence of the light chain variable region ofthe rat anti-FGFR2 antibody FR2-14 (FIG. 34).SEQ ID NO: 27: DNA fragment comprising a DNA sequence encoding the aminoacids of a human κ chain secretory signal sequence and a human κ chainconstant region (FIG. 35).SEQ ID NO: 28: Primer F for a light chain expression vector (FIG. 36).SEQ ID NO: 29: Primer R for a light chain expression vector (FIG. 37).SEQ ID NO: 30: DNA fragment comprising a DNA sequence encoding the aminoacids of a human heavy chain signal sequence and a human IgG1 constantregion (FIG. 38).SEQ ID NO: 31: Nucleotide sequence of the light chain of human chimericFR2-10 (cFR2-10) (FIG. 39). In this sequence, nucleotide positions 1 to60 represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature cFR2-10 light chains.SEQ ID NO: 32: Amino acid sequence of the light chain of human chimericFR2-10 (cFR2-10) (FIG. 40). In this sequence, amino acid positions 1 to20 represent a signal sequence, which is usually not contained in theamino acid sequences of most of mature cFR2-10 light chains.SEQ ID NO: 33: Primer set F for the light chain of human chimeric FR2-10(FIG. 41).SEQ ID NO: 34: Primer set R for the light chain of human chimeric FR2-10(FIG. 42).SEQ ID NO: 35: Nucleotide sequence of the heavy chain of human chimericFR2-10 (cFR2-10) (FIG. 43). In this sequence, nucleotide positions 1 to57 represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature cFR2-10 heavy chains.SEQ ID NO: 36: Amino acid sequence of the heavy chain of human chimericFR2-10 (cFR2-10) (FIG. 44). In this sequence, amino acid positions 1 to19 represent a signal sequence, which is usually not contained in theamino acid sequences of most of mature cFR2-10 heavy chains.SEQ ID NO: 37: Primer set F for the heavy chain of human chimeric FR2-10(FIG. 45).SEQ ID NO: 38: Primer set R for the heavy chain of human chimeric FR2-10(FIG. 46).SEQ ID NO: 39: Nucleotide sequence of the light chain of human chimericFR2-13 (cFR2-13) (FIG. 47). In this sequence, nucleotide positions 1 to60 represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature cFR2-13 light chains.SEQ ID NO: 40: Amino acid sequence of the light chain of human chimericFR2-13 (cFR2-13) (FIG. 48). In this sequence, amino acid positions 1 to20 represent a signal sequence, which is usually not contained in theamino acid sequences of most of mature cFR2-13 light chains.SEQ ID NO: 41: Primer F for the light chain of human chimeric FR2-13(FIG. 49).SEQ ID NO: 42: Primer R for the light chain of human chimeric FR2-13(FIG. 50).SEQ ID NO: 43: Nucleotide sequence of the heavy chain of human chimericFR2-13 (cFR2-13) (FIG. 51). In this sequence, nucleotide positions 1 to57 represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature cFR2-13 heavy chains.SEQ ID NO: 44: Amino acid sequence of the heavy chain of human chimericFR2-13 (cFR2-13) (FIG. 52). In this sequence, amino acid positions 1 to19 represent a signal sequence, which is usually not contained in theamino acid sequences of most of mature cFR2-13 heavy chains.SEQ ID NO: 45: Primer F for the heavy chain of human chimeric FR2-13(FIG. 53).SEQ ID NO: 46: Primer R for the heavy chain of human chimeric FR2-13(FIG. 54).SEQ ID NO: 47: Nucleotide sequence of the light chain of human chimericFR2-14 (cFR2-14) (FIG. 55). In this sequence, nucleotide positions 1 to60 represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature cFR2-14 light chains.SEQ ID NO: 48: Amino acid sequence of the light chain of human chimericFR2-14 (cFR2-14) (FIG. 56). In this sequence, amino acid positions 1 to20 represent a signal sequence, which is usually not contained in theamino acid sequences of most of mature cFR2-14 light chains.SEQ ID NO: 49: Primer for the light chain of human chimeric FR2-14 (FIG.57).SEQ ID NO: 50: Nucleotide sequence of the heavy chain of human chimericFR2-14 (cFR2-14) (FIG. 58). In this sequence, nucleotide positions 1 to57 represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature cFR2-14 heavy chains.SEQ ID NO: 51: Amino acid sequence of the heavy chain of human chimericFR2-14 (cFR2-14) (FIG. 59). In this sequence, amino acid positions 1 to19 represent a signal sequence, which is usually not contained in theamino acid sequences of most of mature cFR2-14 heavy chains.SEQ ID NO: 52: Amino acid sequence of the heavy chain CDR1 of the ratanti-FGFR2 antibody FR2-10 (FIG. 60).SEQ ID NO: 53: Amino acid sequence of the heavy chain CDR2 of the ratanti-FGFR2 antibody FR2-10 (FIG. 61).SEQ ID NO: 54: Amino acid sequence of the heavy chain CDR3 of the ratanti-FGFR2 antibody FR2-10 (FIG. 62).SEQ ID NO: 55: Amino acid sequence of the heavy chain CDR1 of the ratanti-FGFR2 antibody FR2-13 (FIG. 63).SEQ ID NO: 56: Amino acid sequence of the heavy chain CDR2 of the ratanti-FGFR2 antibody FR2-13 (FIG. 64).SEQ ID NO: 57: Amino acid sequence of the heavy chain CDR3 of the ratanti-FGFR2 antibody FR2-13 (FIG. 65).SEQ ID NO: 58: Amino acid sequence of the heavy chain CDR1 of the ratanti-FGFR2 antibody FR2-14 (FIG. 66).SEQ ID NO: 59: Amino acid sequence of the heavy chain CDR2 of the ratanti-FGFR2 antibody FR2-14 (FIG. 67).SEQ ID NO: 60: Amino acid sequence of the heavy chain CDR3 of the ratanti-FGFR2 antibody FR2-14 (FIG. 68).SEQ ID NO: 61: Amino acid sequence of the light chain CDR1 of the ratanti-FGFR2 antibody FR2-10 (FIG. 69).SEQ ID NO: 62: Amino acid sequence of the light chain CDR2 of the ratanti-FGFR2 antibody FR2-10 (FIG. 70).SEQ ID NO: 63: Amino acid sequence of the light chain CDR3 of the ratanti-FGFR2 antibody FR2-10 (FIG. 71).SEQ ID NO: 64: Amino acid sequence of the light chain CDR1 of the ratanti-FGFR2 antibody FR2-13 (FIG. 72).SEQ ID NO: 65: Amino acid sequence of the light chain CDR2 of the ratanti-FGFR2 antibody FR2-13 (FIG. 73).SEQ ID NO: 66: Amino acid sequence of the light chain CDR3 of the ratanti-FGFR2 antibody FR2-13 (FIG. 74).SEQ ID NO: 67: Amino acid sequence of the light chain CDR1 of the ratanti-FGFR2 antibody FR2-14 (FIG. 75).SEQ ID NO: 68: Amino acid sequence of the light chain CDR2 of the ratanti-FGFR2 antibody FR2-14 (FIG. 76).SEQ ID NO: 69: Amino acid sequence of the light chain CDR3 of the ratanti-FGFR2 antibody FR2-14 (FIG. 77).SEQ ID NO: 70: Amino acid sequence of human FGFR2 IIIb (FIG. 78).SEQ ID NO: 71: Amino acid sequence of human FGFR2 IIIc (FIG. 79).SEQ ID NO: 72: Nucleotide sequence of a humanized FR2-14 light chain(hFR2-14_L1) (FIG. 80). In this sequence, nucleotide positions 1 to 60represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature light chains hFR2-14_L1.SEQ ID NO: 73: Amino acid sequence of the humanized FR2-14 light chain(hFR2-14_L1) (FIG. 81). In this sequence, amino acid positions 1 to 20represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature light chains hFR2-14_L1.SEQ ID NO: 74: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H1) (FIG. 82). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H1.SEQ ID NO: 75: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H1) (FIG. 83). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H1.SEQ ID NO: 76: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H2) (FIG. 84). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H2.SEQ ID NO: 77: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H2) (FIG. 85). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H2.SEQ ID NO: 78: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H3) (FIG. 86). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H3.SEQ ID NO: 79: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H3) (FIG. 87). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H3.SEQ ID NO: 80: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H4) (FIG. 88). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H4.SEQ ID NO: 81: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H4) (FIG. 89). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H4.SEQ ID NO: 82: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H5) (FIG. 90). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H5.SEQ ID NO: 83: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H5) (FIG. 91). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H5.SEQ ID NO: 84: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H6) (FIG. 92). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H6.SEQ ID NO: 85: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H6) (FIG. 93). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H6.SEQ ID NO: 86: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H7) (FIG. 94). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H7.SEQ ID NO: 87: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H7) (FIG. 95). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H7.SEQ ID NO: 88: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H8) (FIG. 96). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H8.SEQ ID NO: 89: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H8) (FIG. 97). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H8.SEQ ID NO: 90: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H9) (FIG. 98). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H9.SEQ ID NO: 91: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H9) (FIG. 99). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H9.SEQ ID NO: 92: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H10) (FIG. 100). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H10.SEQ ID NO: 93: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H10) (FIG. 101). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H10.SEQ ID NO: 94: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H11) (FIG. 102). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H11.SEQ ID NO: 95: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H11) (FIG. 103). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H11.SEQ ID NO: 96: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H12 or hFR2-14_H19) (FIG. 104). In this sequence, nucleotidepositions 1 to 57 represent a signal sequence, which is usually notcontained in the nucleotide sequences of most of mature heavy chainshFR2-14_H12 or hFR2-14_H19.SEQ ID NO: 97: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H12 or hFR2-14_H19) (FIG. 105). In this sequence, amino acidpositions 1 to 19 represent a signal sequence, which is usually notcontained in the amino acid sequences of most of mature heavy chainshFR2-14_H12 or hFR2-14_H19.SEQ ID NO: 98: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H13) (FIG. 106). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H13.SEQ ID NO: 99: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H13) (FIG. 107). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H13.SEQ ID NO: 100: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H14) (FIG. 108). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H14.SEQ ID NO: 101: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H14) (FIG. 109). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H14.SEQ ID NO: 102: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H15) (FIG. 110). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H15.SEQ ID NO: 103: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H15) (FIG. 111). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H15.SEQ ID NO: 104: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H16) (FIG. 112). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H16.SEQ ID NO: 105: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H16) (FIG. 113). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H16.SEQ ID NO: 106: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H17) (FIG. 114). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H17.SEQ ID NO: 107: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H17) (FIG. 115). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H17.SEQ ID NO: 108: Nucleotide sequence of a humanized FR2-14 heavy chain(hFR2-14_H18) (FIG. 116). In this sequence, nucleotide positions 1 to 57represent a signal sequence, which is usually not contained in thenucleotide sequences of most of mature heavy chains hFR2-14_H18.SEQ ID NO: 109: Amino acid sequence of the humanized FR2-14 heavy chain(hFR2-14_H18) (FIG. 117). In this sequence, amino acid positions 1 to 19represent a signal sequence, which is usually not contained in the aminoacid sequences of most of mature heavy chains hFR2-14_H18.SEQ ID NO: 110: Primer VH3A-F for an hFR2-14_H2 type heavy chain (FIG.118).SEQ ID NO: 111: Primer VH3A-R for an hFR2-14_H2 type heavy chain (FIG.119).SEQ ID NO: 112: Primer D23fw for gene amplification of D2 (FIG. 120).SEQ ID NO: 113: Primer D23rv for gene amplification of D2 (FIG. 121).

1. An antibody or a functional fragment thereof which has antibodydependent cellular cytotoxic activity and binds to a fibroblast growthfactor receptor (FGFR).
 2. The antibody or functional fragment thereofaccording to claim 1, wherein the fibroblast growth factor receptor(FGFR) is human FGFR.
 3. The antibody or functional fragment thereofaccording to claim 1, wherein the fibroblast growth factor receptor(FGFR) is FGFR2.
 4. The antibody or functional fragment thereofaccording to claim 1, wherein the antibody or functional fragmentthereof binds to human fibroblast growth factor receptor 2 (human FGFR2)IIIb and/or human fibroblast growth factor receptor 2 (human FGFR2)IIIc.
 5. The antibody or functional fragment thereof according to claim1, wherein the antibody or functional fragment thereof binds to humanfibroblast growth factor receptor 2 (human FGFR2) IIIb and humanfibroblast growth factor receptor 2 (human FGFR2) IIIc.
 6. The antibodyor functional fragment thereof according to claim 1, wherein theantibody or functional fragment thereof binds to one or two or moreimmunoglobulin-like domains of the human fibroblast growth factorreceptor
 2. 7. The antibody or functional fragment thereof according toclaim 1, wherein the antibody or functional fragment thereof binds toimmunoglobulin-like domain 2 of the human fibroblast growth factorreceptor
 2. 8. The antibody or functional fragment thereof according toclaim 1, wherein the antibody or functional fragment thereof binds toimmunoglobulin-like domain 3 of the human fibroblast growth factorreceptor
 2. 9. The antibody or functional fragment thereof according toclaim 1, wherein the antibody or functional fragment thereof hasneutralizing activity against the human fibroblast growth factorreceptor 2 (human FGFR2) IIIb and/or the human fibroblast growth factorreceptor 2 (human FGFR2) IIIc.
 10. The antibody or functional fragmentthereof according to claim 1, wherein the antibody or functionalfragment thereof has neutralizing activity against the human fibroblastgrowth factor receptor 2 (human FGFR2) IIIb and the human fibroblastgrowth factor receptor 2 (human FGFR2) IIIc.
 11. The antibody orfunctional fragment thereof according to claim 1, wherein the antibodyor functional fragment thereof has antitumor activity.
 12. The antibodyor functional fragment thereof according to claim 11, wherein theantibody or functional fragment thereof exhibits antitumor activity invivo.
 13. The antibody or functional fragment thereof according to claim1, wherein the antibody comprises a heavy chain comprising CDRH1comprising SEQ ID NO: 52 or an amino acid sequence derived from SEQ IDNO: 52 by the substitution of one or two amino acids; CDRH2 comprisingSEQ ID NO: 53 or an amino acid sequence derived from SEQ ID NO: 53 bythe substitution of one or two amino acids; and CDRH3 comprising SEQ IDNO: 54 or an amino acid sequence derived from SEQ ID NO: 54 by thesubstitution of one or two amino acids; and a light chain comprisingCDRL1 comprising SEQ ID NO: 61 or an amino acid sequence derived fromSEQ ID NO: 61 by the substitution of one or two amino acids; CDRL2comprising SEQ ID NO; 62 or an amino acid sequence derived from SEQ IDNO: 62 by the substitution of one or two amino acids; and CDRL3comprising SEQ ID NO: 63 or an amino acid sequence derived from SEQ IDNO: 63 by the substitution of one or two amino acids, and binds to humanFGFR2.
 14. The antibody or functional fragment thereof according toclaim 1, wherein the antibody comprises a heavy chain comprising CDRH1comprising SEQ ID NO: 55 or an amino acid sequence derived from SEQ IDNO: 55 by the substitution of one or two amino acids; CDRH2 comprisingSEQ ID NO: 56 or an amino acid sequence derived from SEQ ID NO: 56 bythe substitution of one or two amino acids; and CDRH3 comprising SEQ IDNO: 57 or an amino acid sequence derived from SEQ ID NO: 57 by thesubstitution of one or two amino acids; and a light chain comprisingCDRL1 comprising SEQ ID NO: 64 or an amino acid sequence derived fromSEQ ID NO: 64 by the substitution of one or two amino acids; CDRL2comprising SEQ ID NO: 65 or an amino acid sequence derived from SEQ IDNO: 65 by the substitution of one or two amino acids; and CDRL3comprising SEQ ID NO: 66 or an amino acid sequence derived from SEQ IDNO: 66 by the substitution of one or two amino acids, and binds to humanFGFR2.
 15. The antibody or functional fragment thereof according toclaim 1, wherein the antibody comprises a heavy chain comprising CDRH1comprising SEQ ID NO; 58 or an amino acid sequence derived from SEQ IDNO: 58 by the substitution of one or two amino acids; CDRH2 comprisingSEQ ID NO: 59 or an amino acid sequence derived from SEQ ID NO 59 by thesubstitution of one or two amino acids; and CDRH3 comprising SEQ ID NO:60 or an amino acid sequence derived from SEQ ID NO: 60 by thesubstitution of one or two amino acids; and a light chain comprisingCDRL1 comprising SEQ ID NO: 67 or an amino acid sequence derived fromSEQ ID NO: 67 by the substitution of one or two amino acids; CDRL2comprising SEQ ID NO: 68 or an amino acid sequence derived from SEQ IDNO: 68 by the substitution of one or two amino acids; and CDRL3comprising SEQ ID NO: 69 or an amino acid sequence derived from SEQ IDNO: 69 by the substitution of one or two amino acids, and binds to humanFGFR2.
 16. The antibody or functional fragment thereof according toclaim 15, wherein the CDRH3 comprises an amino acid sequence derivedfrom SEQ ID NO: 60 by the substitution of one or two amino acids. 17.The antibody or functional fragment thereof according to claim 1,wherein the antibody is a monoclonal antibody.
 18. The antibody orfunctional fragment thereof according to claim 1, wherein the antibodyis a chimeric antibody.
 19. The antibody or functional fragment thereofaccording to claim 1, wherein the antibody is a humanized antibody. 20.The antibody or functional fragment thereof according to claim 19,wherein the antibody is selected from the following (i) to (xix): (i) ahumanized antibody (hFR2-14_H19/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 97; (ii) ahumanized antibody (hFR2-14_H12/L1) comprising alight chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 97; (iii) ahumanized antibody (hFR2-14_H8/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 89; (iv) ahumanized antibody (hFR2-14_H11/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 95; (v) ahumanized antibody (hFR2-14_H5/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 83; (vi) ahumanized antibody (hFR2-14_H1/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 75); (vii) ahumanized antibody (hFR2-14_H2/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 77; (viii) ahumanized antibody (hFR2-14_H13/L1) comprising a Tight chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 79; (ix) ahumanized antibody (hFR2-14_H4/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 81; (x) ahumanized antibody (hFR2-14_H6/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 85; (xi) ahumanized antibody (hFR2-14_H7/L1) comprising a fight chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 87; (xii) ahumanized antibody (hFR2-14_H9/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 91; (xiii) ahumanized antibody (hFR2-14_H10/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 93; (xiv) ahumanized antibody (hFR2-14_H13/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 99; (xv) ahumanized antibody (hFR2-14_H14/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 101; (xvi) ahumanized antibody (hFR2-14_H15/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 103; (xvii) ahumanized antibody (hFR2-14_H16/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of SEQ ID NO: 105; (xviii) ahumanized antibody (hFR2-14_H17/L1) comprising a light chain comprisingamino acid positions 21 to 235 of SEQ ID NO: 73, and a heavy chaincomprising amino acid positions 20 to 467 of represented by SEQ ID NO:107; and (xix) a humanized antibody (hFR2-14_H18/L1) comprising a lightchain comprising amino acid positions 21 to 235 of SEQ ID NO: 73, and aheavy chain comprising amino acid positions 20 to 467 of SEQ ID NO: 109.21. The antibody or functional fragment thereof according to claim 1,wherein the antibody comprises heavy and light chains comprising aminoacid sequences having 95% or higher identity to the amino acid sequencesof the heavy and light chains, respectively, of an antibody according toclaim 20, and binds to human FGFR2.
 22. The antibody or functionalfragment thereof according to claim 1, wherein the antibody orfunctional fragment thereof binds to a site on an antigen recognized byan antibody according to claim
 20. 23. The antibody or functionalfragment thereof according to claim 1, wherein the antibody orfunctional fragment thereof competes with an antibody according to claim20 for binding to human FGFR2.
 24. The antibody or functional fragmentthereof according to claim 1, wherein the antibody or functionalfragment thereof binds to an epitope on human FGFR2, the epitope beingconstituted by tyrosine (Tyr) at residue 155, threonine (Thr) at residue157, lysine (Lys) at residue 176, alanine (Ala) at residue 181, glycine(Gly) at residue 182, glycine (Gly) at residue 183, asparagine (Asn) atresidue 184, proline (Pro) at residue 185, methionine (Met) at residue186, threonine (Thr) at residue 188, glutamine (Gin) at residue 200,glutamic acid (Glu) at residue 201, glycine (Gly) at residue 205,glycine (Gly) at residue 206, lysine (Lys) at residue 208, valine (Val)at residue 209, arginine (Arg) at residue 210, asparagine (Asn) atresidue 211, glutamine (Gln) at residue 212, histidine (His) at residue213, tryptophan (Trp) at residue 214, and isoleucine (Ile) at residue217 in the amino acid sequence represented by SEQ ID NO:
 70. 25. Theantibody or functional fragment thereof according to claim 1, whereinthe antibody or functional fragment thereof has an interaction distancewith each of tyrosine (Tyr) at residue 155, threonine (Thr) at residue157, lysine (Lys) at residue 176, alanine (Ala) at residue 181, glycine(Gly) at residue 182, glycine (Gly) at residue 183, asparagine (Asn) atresidue 184, proline (Pro) at residue 185, methionine (Met) at residue186, threonine (Thr) at residue 188, glutamine (Gin) at residue 200,glutamic acid (Glu) at residue 201, glycine (Gly) at residue 205,glycine (Gly) at residue 206, lysine (Lys) at residue 208, valine (Val)at residue 209, arginine (Arg) at residue 210, asparagine (Asn) atresidue 211, glutamine (Gin) at residue 212, histidine (His) at residue213, tryptophan (Trp) at residue 214, and isoleucine (Ile) at residue217 in the amino acid sequence represented by SEQ ID NO:
 70. 26. Theantibody or functional fragment thereof according to claim 25, whereinthe interaction distance is 6 angstroms or shorter.
 27. The antibody orfunctional fragment thereof according to claim 25, wherein theinteraction distance is 4 angstroms or shorter.
 28. The antibody orfunctional fragment thereof according to claim 1, wherein the antibodyis a human antibody.
 29. The antibody or functional fragment thereofaccording to claim 1, wherein the antibody or functional fragmentthereof inhibits the binding of FGF to human FGFR2.
 30. The antibody orfunctional fragment thereof according to claim 1, wherein the antibodyor functional fragment thereof has antibody dependent cellular cytotoxicactivity and/or antibody dependent cell phagocytosis activity.
 31. Anucleotide of any one of the following (i) to (iii): (i) a nucleotidecomprising a nucleotide sequence encoding a partial or whole amino acidsequence of the heavy or light chain of an antibody according to claim1; (ii) a nucleotide comprising the nucleotide sequence encoding apartial or whole amino acid sequence of the heavy or tight chain of anantibody according to claim 1; and (iii) a nucleotide consisting of thenucleotide sequence encoding a partial or whole amino acid sequence ofthe heavy or light chain of an antibody according to claim
 1. 32. Arecombinant vector having an insert of a nucleotide according to claim31.
 33. A recombinant cell comprising a nucleotide according to claim31.
 34. A cell producing an antibody or a functional fragment thereofaccording to claim
 1. 35. A method for producing an antibody or afunctional fragment thereof according to claim 1, comprising thefollowing steps (i) and (ii): (i) culturing a cell according to claim33; and (ii) recovering the antibody or functional fragment thereof fromthe cultures obtained in the step (i).
 36. An antibody or functionalfragment thereof obtained by a method according to claim
 35. 37. Theantibody or functional fragment thereof according to claim 1, wherein 1to 5 amino acids are deleted from the amino terminus or carboxylterminus of the heavy or light chain.
 38. A modified form of an antibodyor a functional fragment thereof according to claim
 1. 39. The modifiedform according to claim 38, wherein a sugar chain modification isregulated.
 40. The antibody or a functional fragment thereof accordingto claim 20, wherein the antibody or functional fragment thereof is amodified antibody or functional fragment, wherein a sugar chainmodification is regulated.
 41. A pharmaceutical composition comprisingan antibody or a functional fragment thereof according to claim 1 as anactive ingredient.
 42. The pharmaceutical composition according to claim41, wherein the pharmaceutical composition is an anticancer agent. 43.The pharmaceutical composition according to claim 42, wherein the canceris FGFR2-positive.
 44. A composition for testing or diagnosis of cancer,comprising an antibody or a functional fragment thereof according toclaim
 1. 45. A composition comprising an antibody or a functionalfragment thereof which has human FGFR2 IIIb selectivity or a modifiedform of the antibody or functional fragment.
 46. The compositionaccording to claim 45, wherein the antibody comprises a heavy chaincomprising SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54 and a lightchain comprising SEQ ID NO: 61, SEQ ID 62, or SEQ ID NO:
 63. 47. Thecomposition according to claim 46, wherein the antibody comprises aheavy chain variable region having the amino acid sequence representedby SEQ ID NO: 12 and a light chain variable region having the amino acidsequence represented by SEQ ID NO:
 21. 48. The composition according toclaim 46, wherein the antibody is a chimeric antibody or a rat antibody.49. The composition according to claim 45, wherein the composition isfor detection or assay of human FGFR2 IIIb.
 50. A method for detectingor assaying human FGFR2 IIIb, comprising the step of contacting a testsample with a composition according to claim
 45. 51. A method fordetecting or assaying human FGFR2 IIIc, comprising the following steps(i) to (iii): (i) contacting a test sample with a composition comprisingan antibody or a functional fragment thereof which selectively binds tohuman FGFR2 IIIb and human FGFR2 IIIc, or a modified form of theantibody or functional fragment to detect or assay human FGFR2 IIIb andhuman FGFR2 IIIc in the test sample; (ii) contacting the test samplewith a composition according to claim 45 to detect or assay the humanFGFR2 IIIb in the test sample; and (iii) comparing the results ofdetection or assay in the step (i) with the results of detection orassay in the step (ii) or subtracting the results of detection or assayin the step (ii) from the results of detection or assay in the step (i)to obtain detection or assay results or a value of the human FGFR2IIIcin the test sample.
 52. The method according to claim 50, wherein themethod is for diagnosis or testing of a human FGFR2-positive cancer. 53.A method for identifying an individual in need of determining the statusof FGFR2 in a sample, comprising the following steps: (i) contacting anindividual-derived sample with a composition according to claim 44; and(ii) determining the individual to be positive when human FGFR2 isdetected in the sample.
 54. The method according to claim 53, whereinthe human FGFR2 is human FGFR2 IIIb.
 55. The method according to claim53, wherein the human FGFR2 is human FGFR2 IIIc and human FGFR2 IIIb.56. The composition according to claim 44, wherein the composition isused in a method according to claim
 53. 57. The method according toclaim 53, wherein the individual has cancer or is at risk thereof.
 58. Apharmaceutical composition comprising an antibody or a functionalfragment thereof which has antibody dependent cellular cytotoxicactivity and binds to a FGFR, wherein the pharmaceutical composition isadministered to an individual identified to be positive by a methodaccording to claim
 53. 59. A reagent comprising an antibody or afunctional fragment thereof according to claim
 1. 60. A method foridentifying a substance having antitumor activity, comprising thefollowing steps (i) to (iii): (i) contacting a test substance with aprotein comprising tyrosine (Tyr) at amino acid position 155 toisoleucine (Ile) at amino acid position 217 in the amino acid sequencerepresented by SEQ ID NO: 70; (ii) measuring or determining the distancebetween the substance and each of tyrosine (Tyr) at residue 155,threonine (Thr) at residue 157, lysine (Lys) at residue 176, alanine(Ala) at residue 181, glycine (Gly) at residue 182, glycine (Gly) atresidue 183, asparagine (Asn) at residue 184, proline (Pro) at residue185, methionine (Met) at residue 186, threonine (Thr) at residue 188,glutamine (Gln) at residue 200, glutamic acid (Glu) at residue 201,glycine (Gly) at residue 205, glycine (Gly) at residue 206, lysine (Lys)at residue 208, valine (Val) at residue 209, arginine (Arg) at residue210, asparagine (Asn) at residue 211, glutamine (Gin) at residue 212,histidine (His) at residue 213, tryptophan (Trp) at residue 214, andisoleucine (Ile) at residue 217 in the amino acid sequence representedby SEQ ID NO: 70 in the protein; and (iii) determining the substance tobe positive when the substance has an interaction distance with each ofthe residues.
 61. The method according to claim 60, further comprisingthe following step (iv): (iv) assaying the antitumor activity of thesubstance.
 62. The method according to claim 60, wherein the substanceis an antibody or a functional fragment thereof, or a modified form ofthe antibody or functional fragment, or a peptide.
 63. A method forproducing a substance determined to be positive in step (iii) accordingto claim 60, comprising preparing the substance by a step including generecombination, peptide synthesis, or in vitro translation.
 64. Thepharmaceutical composition according to claim 41, further comprising anadditional drug.
 65. The antibody or functional fragment thereofaccording to claim 1, wherein the antibody or functional fragment isconjugated with an additional compound.
 66. The pharmaceuticalcomposition according to claim 65, wherein the pharmaceuticalcomposition further comprises an additional drug.